Salts of prodrugs of piperazine and substituted piperidine antiviral agents

ABSTRACT

This invention provides for prodrug Compounds I, pharmaceutical compositions thereof, and their use in treating HIV infection. 
                         
wherein:
     X is C or N with the proviso that when X is N, R 1  does not exist;   W is C or N with the proviso that when W is N, R 2  does not exist;   V is C;   E is hydrogen or a pharmaceutically acceptable salt thereof; and   Y is selected from the group consisting of   

     
       
         
         
             
             
         
       
     
     Also, this invention provides for intermediate Compounds II useful in making prodrug Compounds I. 
                         
wherein:
     L and M are independently selected from the group consisting of C 1 -C 6  alkyl, phenyl, benzyl, trialkylsilyl, -2,2,2-trichloroethoxy and 2-trimethylsilylethoxy.

CROSS REFERENCES TO RELATED APPLICATIONS

This continuation application claims the benefit of U.S. Ser. No.13/429,838 filed Mar. 26, 2012, now U.S. Pat. No. 8,461,333, whichclaims the benefit of U.S. Ser. No. 12/767,222 filed Apr. 26, 2010, nowU.S. Pat. No. 8,168,615, which claims the benefit of U.S. Ser. No.11/066,745 filed Feb. 25, 2005, now U.S. Pat. No. 7,745,625, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/635,231filed Dec. 10, 2004 and 60/553,320 filed Mar. 15, 2004, now expired. Theentire teachings of the referenced applications are herein incorporatedby reference in their entirety.

FIELD OF THE INVENTION

This invention provides compounds having drug and bio-affectingproperties, their pharmaceutical compositions and method of use. Inparticular, the invention is concerned with new prodrug derivatives withantiviral activity. More particularly, the present invention relates tocompounds useful for the treatment of HIV and AIDS.

BACKGROUND ART

HIV-1 (human immunodeficiency virus-1) infection remains a major medicalproblem, with an estimated 42 million people infected worldwide at theend of 2002. The number of cases of HIV and AIDS (acquiredimmunodeficiency syndrome) has risen rapidly. In 2002, ˜5.0 million newinfections were reported, and 3.1 million people died from AIDS.Currently available drugs for the treatment of HIV include tennucleoside reverse transcriptase (RT) inhibitors or approved single pillcombinations (zidovudine or AZT (or Retrovir®), didanosine (or Videx®),stavudine (or Zerit®), lamivudine (or 3TC or Epivir®), zalcitabine (orDDC or Hivid®), abacavir succinate (or Ziagen®), Tenofovir disoproxilfumarate salt (or Viread®), Combivir® (contains—3TC plus AZT), Trizivir®(contains abacavir, lamivudine, and zidovudine) and Emtriva®(emtricitabine); three non-nucleoside reverse transcriptase inhibitors:nevirapine (or Viramune®), delavirdine (or Rescriptor®) and efavirenz(or Sustiva®), nine peptidomimetic protease inhibitors or approvedformulations: saquinavir, indinavir, ritonavir, nelfinavir, amprenavir,lopinavir, Kaletra® (lopinavir and Ritonavir), Atazanavir (Reyataz®),Fosamprenavir® and one fusion inhibitor which targets viral gp41 T-20(FUZEON®). Each of these drugs can only transiently restrain viralreplication if used alone. However, when used in combination, thesedrugs have a profound effect on viremia and disease progression. Infact, significant reductions in death rates among AIDS patients havebeen recently documented as a consequence of the widespread applicationof combination therapy. However, despite these impressive results, 30 to50% of patients ultimately fail combination drug therapies. Insufficientdrug potency, non-compliance, restricted tissue penetration anddrug-specific limitations within certain cell types (e.g. mostnucleoside analogs cannot be phosphorylated in resting cells) mayaccount for the incomplete suppression of sensitive viruses.Furthermore, the high replication rate and rapid turnover of HIV-1combined with the frequent incorporation of mutations, leads to theappearance of drug-resistant variants and treatment failures whensub-optimal drug concentrations are present (Larder and Kemp; Gulick;Kuritzkes; Morris-Jones et al; Schinazi et al; Vacca and Condra;Flexner; Berkhout and Ren et al; (Ref 6-14)). Therefore, novel anti-HIVagents exhibiting distinct resistance patterns, and favorablepharmacokinetic as well as safety profiles are needed to provide moretreatment options.

Currently marketed HIV-1 drugs are dominated by either nucleosidereverse transcriptase inhibitors or peptidomimetic protease inhibitors.Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have recentlygained an increasingly important role in the therapy of HIV infections(Pedersen & Pedersen, Ref 15). At least 30 different classes of NNRTIhave been described in the literature (De Clercq, Ref 16) and severalNNRTIs have been evaluated in clinical trials. Dipyridodiazepinone(nevirapine), benzoxazinone (efavirenz) and bis(heteroaryl) piperazinederivatives (delavirdine) have been approved for clinical use. However,the major drawback to the development and application of NNRTIs is thepropensity for rapid emergence of drug resistant strains, both in tissuecell culture and in treated individuals, particularly those subject tomonotherapy. As a consequence, there is considerable interest in theidentification of NNRTIs less prone to the development of resistance(Pedersen & Pedersen, Ref 15). A recent overview of non-nucleosidereverse transcriptase inhibitors: “Perspectives on novel therapeuticcompounds and strategies for the treatment of HIV infection”. hasappeared (Buckheit, reference 99). A review covering both NRTI andNNRTIs has appeared (De Clercq, reference 100). An overview of thecurrent state of the HIV drugs has been published (De Clercq, reference101).

Several indole derivatives including indole-3-sulfones, piperazinoindoles, pyrazino indoles, and 5H-indolo[3,2-b][1,5]benzothiazepinederivatives have been reported as HIV-1 reverse transciptase inhibitors(Greenlee et al, Ref 1; Williams et al, Ref 2; Romero et al, Ref 3; Fontet al, Ref 17; Romero et al, Ref 18; Young et al, Ref 19; Genin et al,Ref 20; Silvestri et al, Ref 21). Indole 2-carboxamides have also beendescribed as inhibitors of cell adhesion and HIV infection (Boschelli etal, U.S. Pat. No. 5,424,329, Ref 4). 3-Substituted indole naturalproducts (Semicochliodinol A and B, didemethylasterriquinone andisocochliodinol) were disclosed as inhibitors of HIV-1 protease(Fredenhagen et al, Ref 22).

Structurally related aza-indole amide derivatives have been disclosedpreviously (Kato et al, Ref 23(a); Levacher et al, Ref 23(b); Dompe Spa,WO-09504742, Ref 5(a); SmithKline Beecham PLC, WO-09611929, Ref 5(b);Schering Corp., US-05023265, Ref 5(c)). However, these structures differfrom those claimed herein in that they are monoaza-indole mono-amiderather than oxoacetamide derivatives, and there is no mention of the useof these compounds for treating viral infections, particularly HIV.

New drugs for the treatment of HIV are needed for the treatment ofpatients who become resistant to the currently approved drugs describedabove which target reverse transcriptase or the protease. One approachto obtaining these drugs is to find molecules which inhibit new anddifferent targets of the virus. A general class of inhibitors which areunder active study are HIV entry inhibitors. This general classificationincludes drugs aimed at several targets which include chemokine receptor(CCR5 or CXCR4) inhibitors, fusion inhibitors targeting viral gp41, andinhibitors which prevent attachment of the viral envelope, gp120, theits human cellular target CD4. A number of reviews or general papers onviral entry inhibitors have recently appeared and some selectedreferences are:

-   Chemokine receptor antagonists as HIV entry inhibitors. Expert    Opinion on Therapeutic Patents (2004), 14(2), 251-255.-   Inhibitors of the entry of HIV into host cells. Meanwell, Nicholas    A.; Kadow, John F. Current Opinion in Drug Discovery & Development    (2003), 6(4), 451-461.-   Virus entry as a target for anti-HIV intervention. Este, Jose A.    Retrovirology Laboratory irsiCaixa, Hospital Universitari Germans    Trias i Pujol, Universitat Autonoma de Barcelona, Badalona, Spain.    Current Medicinal Chemistry (2003), 10(17), 1617-1632.-   New antiretroviral agents. Rachline, A.; Joly, V. Service de    Maladies Infectieuses et Tropicales A, Hopital Bichat-Claude    Bernard, Paris, Fr. Antibiotiques (2003), 5(2), 77-82.-   New antiretroviral drugs. Gulick, R. M. Cornell HIV Clinical Trials    Unit, Division of International Medicine and Infectious Diseases,    Weill Medical College of Cornell University, New York, N.Y., USA.    Clinical Microbiology and Infection (2003), 9(3), 186-193.-   Sensitivity of HIV-1 to entry inhibitors correlates with    envelope/coreceptor affinity, receptor density, and fusion kinetics.    Reeves, Jacqueline D.; Gallo, Stephen A.; Ahmad, Navid; Miamidian,    John L.; Harvey, Phoebe E.; Sharron, Matthew; Pohlmann, Stefan;    Sfakianos, Jeffrey N.; Derdeyn, Cynthia A.; Blumenthal, Robert;    Hunter, Eric; Doms, Robert W. Department of Microbiology, University    of Pennsylvania, Philadelphia, Pa., USA. Proceedings of the National    Academy of Sciences of the United States of America (2002), 99(25),    16249-16254. CODEN: PNASA6 ISSN: 0027-8424.-   Opportunities and challenges in targeting HIV entry. Biscone, Mark    J.; Pierson, Theodore C.; Doms, Robert W. Department of    Microbiology, University of Pennsylvania, Philadelphia, Pa., USA.    Current Opinion in Pharmacology (2002), 2(5), 529-533.-   HIV entry inhibitors in clinical development. O'Hara, Bryan M.;    Olson, William C. Progenies Pharmaceuticals, Inc., Tarrytown, N.Y.,    USA. Current Opinion in Pharmacology (2002), 2(5), 523-528.-   Resistance mutation in HIV entry inhibitors. Hanna, Sheri L.; Yang,    Chunfu; Owen, Sherry M.; Lal, Renu B. HIV Immunology and Diagnostics    Branch, Division of AIDS, STD, Atlanta, Ga., USA. AIDS (London,    United Kingdom) (2002), 16(12), 1603-1608.-   HIV entry: are all receptors created equal? Goldsmith, Mark A.;    Doms, Robert W. Genencor International, Inc., Palo Alto, Calif.,    USA. Nature Immunology (2002), 3(8), 709-710. CODEN: NIAMCZ ISSN:    1529-2908.-   Peptide and non peptide HIV fusion inhibitors. Jiang, Shibo; Zhao,    Qian; Debnath, Asim K. The New York Blood Center, Lindsley F.    Kimball Research Institute, New York, N.Y., USA. Current    Pharmaceutical Design (2002), 8(8), 563-580.

There are two general approaches for preventing the initial attachmentof viral membrane, gp120, to cellular CD4 which are a) inhibitors whichbind to human CD4 and block attachment of viral envelope (gp120) and b)inhibitors which bind to viral gp120 and prevent the binding of cellularCD4. The second approach has the advantage that it inhibits a viraltarget and, if selective, minimizes the chances of perturbing normalhuman physiology or causing side effects. With this approach, in orderto overcome a spectrum in susceptability to drug caused by variabilityin the sequences of viral envelope and to suppress the development ofresistance, it is important to achieve plasma levels of drug that is asmany multiples as possible over the EC50 or other measure of theconcentration of drug needed to kill virus. As discussed later, theseinhibitors appear safe so to be of wide utility in man they, therefore,must be able to achieve exposure levels sufficient to enable virussuppression. The higher the multiple of drug levels over the levelneeded to inhibit viral growth, the more efficiently and completely thesuppression of viral replication and the lower the chance for viralmutation and subsequent development of resistance to treatment. Thus,important aspects contributing to the efficacy of viral attachmentinhibitors include not only intrinsic potency and safety, but alsopharmacokinetics and pharmaceutical properties which allow attainment ofhigh plasma exposure at a physically feasible dose and an acceptable,preferably convenient, administration schedule. This invention describesa prodrug approach which greatly enhances the maximum exposure and theability to increase exposure multiples (i.e., multiples of drug exposuregreater than EC₅₀ or EC₉₀) upon dose escalation of efficacious membersof a previously disclosed class of HIV attachment inhibitors.

A series of recent publications and disclosures characterize anddescribe a compound labelled as BMS-806, an initial member of a class ofviral entry inhibitors which target viral gp-120 and prevent attachmentof virus to host CD4.

-   A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and    inhibits CD4 receptor binding. Lin, Pin-Fang; Blair, Wade; Wang,    Tao; Spicer, Timothy; Guo, Qi; Zhou, Nannan; Gong, Yi-Fei; Wang,    H.-G. Heidi; Rose, Ronald; Yamanaka, Gregory; Robinson, Brett; Li,    Chang-Ben; Fridell, Robert; Deminie, Carol; Demers, Gwendeline;    Yang, Zheng; Zadjura, Lisa; Meanwell, Nicholas; Colonno, Richard.    Proceedings of the National Academy of Sciences of the United States    of America (2003), 100(19), 11013-11018.-   Biochemical and genetic characterizations of a novel human    immunodeficiency virus type 1 inhibitor that blocks gp120-CD4    interactions. Guo, Qi; Ho, Hsu-Tso; Dicker, Ira; Fan, Li; Zhou,    Nannan; Friborg, Jacques; Wang, Tao; McAuliffe, Brian V.; Wang,    Hwei-gene Heidi; Rose, Ronald E.; Fang, Hua; Scarnati, Helen T.;    Langley, David R.; Meanwell, Nicholas A.; Abraham, Ralph; Colonno,    Richard J.; Lin, Pin-fang. Journal of Virology (2003), 77(19),    10528-10536.-   Method using small heterocyclic compounds for treating HIV infection    by preventing interaction of CD4 and gp120. Ho, Hsu-Tso; Dalterio,    Richard A.; Guo, Qi; Lin, Pin-Fang. PCT Int. Appl. (2003), WO    2003072028A2.-   Discovery of    4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine    (BMS-378806): A Novel HIV-1 Attachment Inhibitor That Interferes    with CD4-gp120 Interactions. Wang, Tao; Zhang, Zhongxing; Wallace,    Owen B.; Deshpande, Milind; Fang, Haiquan; Yang, Zheng; Zadjura,    Lisa M.; Tweedie, Donald L.; Huang, Stella; Zhao, Fang; Ranadive,    Sunanda; Robinson, Brett S.; Gong, Yi-Fei; Ricarrdi, Keith; Spicer,    Timothy P.; Deminie, Carol; Rose, Ronald; Wang, Hwei-Gene Heidi;    Blair, Wade S.; Shi, Pei-Yong; Lin, Pin-fang; Colonno, Richard J.;    Meanwell, Nicholas A. Journal of Medicinal Chemistry (2003), 46(20),    4236-4239.

Indole, azaindole and other oxo amide containing derivatives from thisclass have been disclosed in a number of different PCT and issued U.S.patent applications (Reference 93-95, 106, 108, 109, 110, 111, and 112)and these references directly relate to the compounds in this patentapplication. None of the compounds in these references of prior artcontain a methyl dihydrogen phosphate (or salt or mono or di ester ofthe phosphate group) group appended to the N-1 nitrogen and thus thecompounds of this current invention represent new compositions ofmatter. This moiety dramatically increases the utility of the parentcompounds by functioning as a prodrug modification which dramaticallyincreases the maximum systemic exposure of the parent molecules inpreclinical models of human exposure. We believe nothing in the priorart references can be construed to disclose or suggest the novelcompounds of this invention and their use to inhibit HIV infection.

This invention describes prodrugs of specific indole and azaindoleketopiperazine amides which are extremely effective at improving theoral utility of the parent molecules as antiviral agents particularly asanti HIV drugs. The parent molecules are relatively insoluble, andsuffer from dissolution-limited or solubility limited absorption whichmeans as the dose is increased above a maximum level, less and less ofthe drug dissolves in time to be absorbed into the circulation and theyare instead passed through the body to be eliminated as waste. Theimprovements offered by the prodrug are necessary, for they allow druglevels in the body to be increased significantly, which provides greaterefficacy vs HIV virus and in particular vs less sensitive or moreresistant strains. Prodrugs are especially important for this class ofdrugs since the drugs target the envelope of the HIV virus, a targetwhich varies from strain to strain and thus in which maximum exposuremultiples are desired. Because with a prodrug, more of the drug will beabsorbed and reach the target, pill burden, cost to the patient anddosing intervals could be reduced. The identification of prodrugs withthese properties is difficult and neither straightforward, nor is aclear path to successful prodrug design disclosed in the literature.There is no clear prior art teaching of which prodrug chemistry toemploy nor which will be most effective. The following discussion anddata will show that the prodrugs described in this invention worksurprisingly well. They release parent drug extremely quickly andefficiently and enhance the exposure to levels which are higher thanreported for many prodrugs.

The use of prodrug strategies or methodologies to markedly enhanceproperties of a drug or to overcome an inherent deficiency in thepharmaceutic or pharmacokinetic properties of a drug can be used incertain circumstances to markedly enhance the utility of a drug.Prodrugs differ from formulations in that chemical modifications lead toan entirely new chemical entity which upon administration to thepatient, regenerates the parent molecule within the body. A myriad ofprodrug strategies exist which provide choices in modulating theconditions for regeneration of the parent drug, the physical,pharmaceutic, or pharmacokinetic properties of the prodrug, and thefunctionality to which the prodrug modifications may be attached.However, none of these publications teach what approach to use thatresult in the specific prodrugs herein invented. A number of reviews ordiscussions on prodrug strategies have been published and anonexhaustive list is provided below:

-   Hydrolysis in Drug and prodrug Metabolism. Richard Testa and Joachim    Mayer, 2003 Wiley-VCH publisher, ISBN 3-906390-25-x.-   Design of Prodrugs, Bundgard, H. Editor, Elsevier, Amsterdam, 1985.-   Pharmacokinetics of drug targeting: specific implications for    targeting via prodrugs. Stella, V. J.; Kearney, A. S. Dep. Pharm.    Chem., Univ. Kansas, Lawrence, Kans., USA. Handbook of Experimental    Pharmacology (1991), 100 (Targeted Drug Delivery), 71-103. CODEN:    HEPHD2 ISSN: 0171-2004. Journal; General Review written in English.    CAN 116:158649 AN 1992:158649 CAPLUS (Copyright 2004 ACS on    SciFinder (R)).-   Prodrugs. Do they have advantages in clinical practice? Stella, V.    J.; Charman, W. N. A.; Naringrekar, V. H. Dep. Pharm. Chem., Univ.    Kansas, Lawrence, Kans., USA. Drugs (1985), 29(5), 455-73. CODEN:    DRUGAY ISSN: 0012-6667. Journal; General Review written in English.    CAN 103:115407 AN 1985:515407 CAPLUS (Copyright 2004 ACS on    SciFinder (R)).-   Trends in prodrug research. Stella, V. J.; Naringrekar, V. H.;    Charman, W. N. A. Dep. Pharm. Chem., Univ. Kansas, Lawrence, Kans.,    USA. Pharmacy International (1984), 5(11), 276-9. CODEN: PHINDQ    ISSN: 0167-3157. Journal; General Review written in English. CAN    102:72143 AN 1985:72143 CAPLUS (Copyright 2004 ACS on SciFinder    (R)).

While some technologies are known to have specific applications, ie toimprove solubility or absorption for example, the development ofprodrugs remains, to a great extent, an empirical exercise. Thus anumber of strategies or chemical modifications must usually be surveyedand the resulting compounds evaluated in biological models in order toascertain and gauge the success of prodrug strategies.

A successful prodrug strategy requires that a chemically reactive sitein a molecule be modified via addition of the prodrug moiety and thatlater under the desired conditions in the patients the prodrug moietywill unmask and release parent drug. The prodrug molecule must havesuitable stability in an acceptable dosage form prior to dosing. Inaddition, the release mechanism must allow the prodrug to regenerateparent drug efficiently and with kinetics that provide therapeuticlevels of parent drug at the disease target. In our molecules, theindole or azaindole nitrogen represents an acceptable point ofattachment for a prodrug moiety.

The suggestion that a phosphate group joined by an appropriate chemistryor linker can enhance oral exposure of a parent drug is a concept knownin the art. However, as will be discussed below, it is unpredictable toknow that if using a phosphate group to create a prodrug will work witha given drug substance. The phosphate group temporarily alters thephysical properties of the drug and is, thus, a prodrug which increasesthe aqueous solubility of the resulting molecule, until it is cleaved byalkaline phosphatase in the body or other chemical reaction of arationally designed linker. For example, in the following reference, theauthors conclude phosphates may improve oral efficacy. In this referencea phosphate derivative of an alcohol group in a poorly water soluble,lipophilic drug displayed better oral bioavailbility than two otherprodrugs and appeared to offer an advantage over the parent moleculewhich possessed low oral bioavailability.

-   Evaluation of a targeted prodrug strategy to enhance oral absorption    of poorly water-soluble compounds. Chan, O. Helen; Schmid, Heidi L.;    Stilgenbauer, Linda A.; Howson, William; Horwell, David C.; Stewart,    Barbra H. Pharmaceutical Research (1998), 15(7), 1012-1018.

Two very pertinent and recent papers have published which discuss thedifficulties of identifying phosphate prodrugs with significantadvantages over the parent molecule for oral use.

A paper entitled “Absorption Rate Limit Considerations for OralPhosphate Prodrugs” by Tycho Heimbach et. al. in Pharmaceutical Research2003, Vol 20, No. 6 pages 848-856 states “The surprising inability touse phosphate prodrugs by the oral route prompted a study in a systembeing used to screen drug candidates for absorption potential.” Thispaper also reviews the reasons many phosphate prodrugs were unsuitablefor oral use and discusses several potential rate limiting factors inthe drug absorption process. The paper also identifies the fewsuccessful applications. The paper attempts to identify properties whichmay make some drugs suitable for oral delivery as phosphate prodrugs butthe message is clear that this is still an empirical science. This isemphasized by the conclusions of a second paper by the same authorsentitled “Enzyme mediated precipitation of parent drugs from theirphosphate prodrugs” by Tycho Heimbach et. al in International Journal ofPharmaceutics 2003, 261, 81-92. The authors state in the Abstract thatmany oral phosphate prodrugs have failed to improve the rate or extentof absorption compared to their insoluble parent drugs. Rapid parentdrug generation via intestinal alkaline phosphatase can result insupersaturated solutions, leading to parent drug precipitation. Thiswould limit utility of the oral phopshates. The conclusions of thispaper state (quoted) “In summary, precipitation of parent drugs fromphosphate prodrugs can be enzyme mediated. Preciptitation of certaindrugs can also be observed for certain drugs in the Caco-2 model. Sinceinduction times decrease and nucleation times increase with highsupersaturation ratios, parent drugs can precipitate when targetedprodrugs concentration are much higher than the parent drug's solubilityie for parent drugs with high supersaturation ratios. The extent towhich a parent drug precipitates during conversion of the prodrug isdependent on the prodrug to parent conversion rates, prodrug effect onthe precipitation of parent drug, and the solubilization of the parentdrug.” As can be seen by the author's conclusion, the process is acomplex one and is dependent on many factors which are impossible topredict in advance such as supersaturation ratios, rate of prodrugconversions in vivo, and ability of the intestinal milieu to solubilizeparent and prodrug mixtures.

The two references by Heimbach describe the clinical status of phosphateprodrugs and discuss the many failures and few successful examples. Oneexample of a clinical failure and one example of a success are providedbelow:

Etoposide® is an anticancer drug which is administered either via iv ororal routes. Etoposide phosphate prodrugs are used clinically, but thesestructures differ from the derivatives of the current application asthis prodrug contains a phosphate formed by direct attachment to aphenol moiety of the parent drug. The main reasons for preparing aphosphate prodrug of the drug etoposide were to improve intravenous usevia increased solubility and reduction of excipients. Although thephosphate prodrug was evaluated orally both preclinically and clinicallyit is only used clinically for iv administration.

-   Synthesis of etoposide phosphate, BMY-40481: a water-soluble    clinically active prodrug of etoposide. Saulnier, Mark G.; Langley,    David R.; Kadow, John F.; Senter, Peter D.; Knipe, Jay O.; Tun, Min    Min; Vyas, Dolatrai M.; Doyle, Terrence W. Bristol-Myers Squibb Co.,    Wallingford, Conn., USA. Bioorganic & Medicinal Chemistry Letters    (1994), 4(21), 2567-72 and references therein.

As can be seen from the following two references, the benefits of thephosphate moiety for oral dosing were not clear.

-   Randomized comparison of etoposide pharmacokinetics after oral    etoposide phosphate and oral etoposide. De Jong, R. S.; Mulder, N.    H.; Uges, D. R. A.; Kaul, S.; Winograd, B.; Sleijfer, D. Th.;    Groen, H. J. M.; Willemse, P. H. B.; van der Graaf, W. T. A.; de    Vries, E. G. E. Department of Medical Oncology, University Hospital    Groningen, Groningen, Neth. British Journal of Cancer (1997),    75(11), 1660-1666. This paper compared parent and prodrug directly    and concluded that oral etoposide phosphate does not offer a    clinically relevant benefit over oral etoposide.-   Etoposide bioavailability after oral administration of the prodrug    etoposide phosphate in cancer patients during a phase I study.    Chabot, G. G.; Armand, J.-P.; Terref, C.; De Formi, M.; Abigerges,    D.; Winograd, B.; Igwemezie, L.; Schacter, L.; Kaul, S.; et al.    Department Medicine, Gustave-Roussy Institute, Villejuif, Fr.    Journal of Clinical Oncology (1996), 14(7), 2020-2030.

This earlier paper found that compared with literature data, oral EP hada 19% higher F value compared with oral E either at low or high doses.They concluded this higher F in E from oral prodrug EP appears to be apharmacological advantage that could be of potential pharmacodynamicimportance for this drug. However the previously mentioned study whichreached opposite conclusions was done later and it appears that thedirect comparison data was more valid. Thus adding a phosphate group toimprove solubility is not a guarantee of improved oral efficacy.

A phosphate prodrug of the HIV protease inhibitor Amprenavir wasprepared and is the active ingredient of what has now become an improveddrug for oral use. This is an example of a rare success from thisapproach. The phosphate is directly attached to a hydroxy moiety andserves to enhance solubility. Fosamprenavir alone or in combination withanother protease inhibitor ritonavir, which serves to inhibit CytochromeP450 3A4-mediated metabolic deactivation, allow patients to receivefewer pills, smaller pills (due to the need for less excipients), and toemploy a less frequent dosing schedule. Clearly, the structure ofAmprenavir is significantly different than the molecules of the presentinvention and does not predict success with other classes of drugs orphosphate linker chemistry. Two references on Fosamprenavir are includedbelow but most recent data can be found by searching a database wellknown in the art such as IDDB (A commercial database calledInvestigational Drugs Database produced by Current Drugs Ltd.).

-   Fosamprenavir vertex Pharmaceuticals/GlaxoSmithKline. [Erratum to    document cited in CA138:130388]. Corbett, Amanda H.; Kashuba,    Angela D. M. School of Pharmacy, The University of North Carolina    Hospitals, Chapel Hill, N.C., USA. Current Opinion in    Investigational Drugs (PharmaPress Ltd.) (2002), 3(5), 824.-   Fosamprenavir Vertex Pharmaceuticals/GlaxoSmithKline. Corbett,    Amanda H.; Kashuba, Angela D. M. School of Pharmacy, The University    of North Carolina Hospitals, Chapel Hill, N.C., USA. Current Opinion    in Investigational Drugs (PharmaPress Ltd.) (2002), 3(3), 384-390.

Searching the literature for examples which can be found listed underkeywords “prodrugs of indoles” or “prodrugs of azaindoles” identify anumber of references that have been described. We are not aware of anyreferences in which azaindole prodrugs were prepared via use of a methyldihydrogen phosphate (or salt or mono or di ester of the phosphategroup) moiety attached to N-1.

Regarding indole phosphate prodrugs, the publication by Zhu et. al.describes a study to find an effective phosphate prodrug of PD 154075.In this molecule, either the direct indole phosphate or a methyldihydrogen phosphate or salt prodrug of the indole nitrogen wereunsuitable prodrugs due to a slow rate of regenerating the parentmolecule. Thus the novel and complex linker depicted below was developedto incorporate a solubilizing phosphate.

-   Phosphate prodrugs of PD 154075. Zhu, Zhijian; Chen, Huai-Gu; Goel,    Om P.; Chan, O. Helen; Stilgenbauer, Linda A.; Stewart, Barbra H.    Division of Warner-Lambert Company, Chemical Development,    Parke-Davis Pharmaceutical Research, Ann Arbor, Mich., USA.    Bioorganic & Medicinal Chemistry Letters (2000), 10(10), 1121-1124.

Many of the references describe the design of prodrugs for purposesother than overcoming dissolution-limited absorption. A number of theprodrugs are not attached to the indole or azaindole nitrogen or aredesigned to release radical intermediates rather than the parent drug.The prodrugs described in this art and their properties do not provideobvious solutions for improving the properties of the parent HIVattachment inhibitors.

It has now been found that new methyl dihydrogen phosphate produgs andpharmaceutically acceptable salts of the general structure shown beloware useful as anti HIV agents with a new mechanism that is currently notemployed by existing drugs. The need for drugs with new mechanisms isgreat since patients are left with no options if they become resistantto the current drug classes. In addition, drugs with new mechanisms canbe used in combinations with known classes of inhibitors to cover theemergence of resistance to these drugs since strains of resistant virusare likely still susceptible to drugs with an alternative mechanism.

We have found that these prodrugs are more water soluble than the parentmolecules, and rapidly convert to the parents after oral dosing inrodents or in in vitro assays with human enzymes or tissues. Inaddition, in one oral dose escalation study, a prodrug providedsurprising enhancements in drug exposure (AUC) and maximum concentration(Cmax) as the dose increased. These predictive studies suggest theseprodrugs should provide advantages in dogs and humans.

The parent compound IVa has been studied in human clinical trials. Thecompound was dosed in healthy human volunteers. A graph of the exposurevs dose is shown in FIG. 1.

As can be seen, single doses of a capsule formulation (red triangles)ranged in size from 200-2400 mgs in 200 mg increments. It is alsoreadily apparent from the oral AUCs, that increases in drug exposureincreased much more slowly and less than proportionally with dose. Infact the differences or increase in exposure above 800 mgs is minimalWith the dose ratios of 1:2:4:6:9:12 for capsule treatment under fastedcondition, the ratios of mean Cmax and AUC values are1:1.3:2.4:2.3:2.1:2.7 and 1:1.5:2.3:2.0:1.9:2.4, respectively. Between200-800 mg dose range the increases in systemic exposure is dose-relatedalthough less than dose-proportional, while such exposure is doseindependent above dose of 800 mg. This phenomenon indicates that theabsorption of Compound IVa with the capsule formulation used issaturable under fasted conditions.

The dose proportionality in systemic exposure seems to be much betterpresented under fed condition (high fat meal) as ratios of Cmax and AUCare 1.6 and 1.5, respectively, when the dose ratio is 1:2.3 (800 mg vs.1800 mg). As can be seen by comparing the single 200 mg dose of asolution of IVa (dark red square) with that of the 200 mg capsule dose,exposure from the solution was higher. Dosing with a solution increasedCompound IVa exposure. The Cmax and AUC of the solution wereapproximately 8- and 3-fold, respectively, of those of the capsule (200mg). The relative bioavailability (32%) of the capsule to the solutionformulation suggests absorption is dissolution rate-limited, suggestinga potential to enhance systemic exposure by improving the formulation.

A high fat meal had a positive food-effect on the compound. The Cmaxafter a fed treatment were approximately 2.6 and 4.6 fold for 800 and1800 mg doses, respectively, of those of the fast treatment. The AUCsafter a fed treatment were approximately 2.5 and 4.7 fold for 800 and1800 mg doses, respectively, of those of the fasted treatment. Therelative bioavailability (fed vs. fasted) values were 293% and 509% for800 mg and 1800 mg doses, respectively. The median Tmax changed from1.25 or 2 (fasted) to 4 (fed) hours.

For the 800 mg capsule with food, the average plasma concentration is1001 and 218 ng/mL at 8 and 12 hours post dose, respectively. Theresults supported a q12h or q8h dosing regimen for a targeted Cmin valueof at least 200 ng/mL after multiple doses. This value was selectedbased on preclinical data. A further summary of some of this datapresented as a bar graph is shown in the second bar graph in FIG. 2B.

Multiple Dose Study in Healthy Humans

A placebo-controlled, ascending multiple-dose study to evaluate thesafety, tolerability and pharmacokinetics of Compound IVa in healthyhuman subjects was carried out. Dosing was continued at 12 h intervalsfor 14 days. In summary, the preliminary PK results indicated that,after single and multiple Q12H doses of Compound IVa, the exposure isgenerally dose proportional over the dose ranges of 400 to 1200 mg and400 to 800 mg with high fat meal and light meal, respectively, theexposure seems to be dose independent above these dose levels with thedifferent meal types, the accumulation is low to moderate (up to ˜1.5fold), and that there is a diurnal variation in the exposure in thatexposure is higher after an evening dose than that after a morning dose.Thus, the exposure was better when dosing was combined with a high fatmeal and exposure increases with dose were higher with a high fat meal.

This is similar to the results obtained in the above single dose study.

Multiple Dose Study in HIV Patients

Based on the exposure data from the studies in normal volunteers, anefficacy study was carried out in HIV patients. An initial disclosure ofthis data has been made in a talk and published abstract. “AntiviralActivity, Safety, and Tolerability of a Novel, Oral Small-Molecule HIV-1Attachment Inhibitor, IVa, in HIV-1-Infected Subjects” G. Hanna, J.Lalezari, J. Hellinger, D. Wohl, T. Masterson, W. Fiske, J. Kadow, P-F.Lin, M. Giordano, R. Colonno, D. Grasela. Abstract J-32, Feb. 11, 2004,11th Conference on Retroviruses and Opportunistic Infections (CROI), SanFrancisco, Calif. The study design included HIV+ adults who were eitherantiretroviral therapy naïve or off antiretroviral therapy for ≧16weeks. Their CD4 counts were required to be ≧250 cells/mm3 and plasmaHIV-1 RNA needed to be in the range 5,000-500,000 c/mL. There were 15subjects in each dose arm and the ratio of patients receivingdrug:placebo was 4:1.

The placebo-controlled, sequential study of IVa utilized an initial dosearm of 800 mg PO every 12 h followed by a second dosing arm of 1800 mgPO administered every 12 h. It is important to note that drug wasadministered in a capsule and in combination with a high fat meal toincrease exposure and plasma levels. Study drug was administered for 7days and the morning of Day 8. Subjects were followed for 14 days.

Study Results (for 18 of the 24 Patients Receiving Drug IVa) CompoundIVa Placebo Day 8 Change in Mean (SD) −0.72 (0.51) −0.02 (0.40) HIV RNARange +0.34 to −1.37 +0.45 to −0.26 Over 14 days, log10 c/mL Maximalchange in Mean (SD) −1.00 (0.50) −0.30 (0.08) HIV RNA Range −0.32 to−1.60 −.22 to −0.38 Over 14 days, log10 c/mL Day 8 Change in CD4, Mean(SD)  106 (151)  6 (57) cells/mm3 Range −214 to +272 −35 to +47 MaximalChange in >0.5 log10     8 (67%) 0 HIV RNA Over 14 c/mL days n (%) >1.0log10     7 (58%) 0 c/mL >1.5 log10     3 (25%) c/mL1. As can be seen by the data for the 800 mg dosing level in combinationwith a high fat meal, significant antiviral activity was observed.However, only 58% of patients had >1.0 log drop in viral load. A morerobust antiviral response was seen with the 1800 mg dosing regimen witha high fat meal where the mean response was a −0.96 log 10 drop in viralload. This data shows that this drug has significant antiviral activityat doses of 800 mg and 1800 mg (and thus in between) every 12 hours incombination with a high fat meal and therefore it could play asignificant role in combination therapy. An updated summary of theresults with BMS-043 in man can be obtained by viewing the abstract orslides from the oral presentation: “Antiviral Activity, Safety, andTolerability of a Novel, Oral Small-Molecule HIV-1 Attachment Inhibitor,BMS-488043, in HIV-1-Infected Subjects” G. Hanna, J. Lalezari, J.Hellinger, D. Wohl, T. Masterson, W. Fiske, J. Kadow, P-F. Lin, M.Giordano, R. Colonno, D. Grasela. Abstract J-32, Feb. 11, 2004, 11thConference on Retroviruses and Opportunistic Infections (CROI), SanFrancisco, Calif.

Unfortunately, it will be unfeasible to deliver this drug chronicallyover many months involving administration of a total of 9 capsules of200 mg each, twice a day in combination with a high fat meal for obvioushealth reasons so a new formulation will be needed which providesincreased exposure from a lower dose and which eliminates the need for ahigh fat meal.

Thus the clinical data shows that a method for improving the exposure ofthis drug from lower doses and in the absence of a high fat meal isnecessary.

Thus the initial data on the prodrugs of this invention surprisinglypredicts they will improve the exposure of molecules such as IVa anddeliver the parent drugs in concentrations that will allow the drugs tobe used in the absence of high fat meals, with lower capsule burden, andchronically as a component of antiretroviral therapy.

Initial data obtained from dosing solid capsules of either the prodrugIab (lysine salt) or solid parent molecule (IVa) to dogs are summarizedin the first bar graph FIG. 2A of FIG. 2. As can be seen, after dosingprodrug Iab (mono lysine salt) either in fasted or dogs fed a high fatmeal, the exposure is surprisingly high when compared to dosing parentmolecule. Also, the effect of fed vs fasted state is minimal if any forthe prodrug yet has an obvious effect on the exposure after dosing thesolid parent molecule. Thus, surprisingly, the exposure of parentmolecule after dosing of prodrug, shows no dependence on a high fat mealwhich predicts for more consistent exposure levels after dosing thanthose from parent molecule. The bar graph in FIG. 2B summarizes thepreviously discussed human data for parent molecule IVa and shows thedependence of exposure for the molecule on the high fat meal, the nonproportional increases in exposure vs dose, and the better exposure fromdosing a solution rather than solid formulation. As discussed below,this shows dissolution or absorption rate limited exposure which inpreclinical models, appears to be surprisingly improved via use of thephosphate prodrug. The use of phosphate prodrugs also increased exposurein fasted dogs vs parent for two other prodrugs. Details of theseexperiments are contained in the experimental section. In addition,details from various studies in rats, dogs, and monkeys for twoadditional prodrug examples and in rats for another two prodrugsdemonstrate the surprising utility of these prodrugs to increaseexposure over that obtained parent molecule at doses which correlate tothose likely to be of utility for the treatment and inhibition of HIVviral replication.

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SUMMARY OF THE INVENTION

The present invention comprises compounds of Formula I, theirpharmaceutical formulations, and their use in patients suffering from orsusceptible to a virus such as HIV. The compounds of Formula I, whichinclude nontoxic pharmaceutically acceptable salts thereof, have theformula and meaning as described below.

The present invention comprises a compound of Formula I,

wherein:X is C or N with the proviso that when X is N, R¹ does not exist;W is C or N with the proviso that when W is N, R² does not exist;V is C;R¹ is hydrogen, methoxy or halogen;R² is hydrogen;R³ is methoxy or heteroaryl, each of which may be independentlyoptionally substituted with one substituent selected from G; whereinheteroaryl is triazolyl, pyrazolyl or oxadiazolyl;E is hydrogen or a pharmaceutically acceptable mono or bis salt thereof;Y is selected from the group consisting of

R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ are each independently H ormethyl, with the proviso that not more than two of R¹⁰-R¹⁷ are methyl;R¹⁸ is selected from the group consisting of C(O)-phenyl,C(O)-pyridinyl, pyridinyl, pyrimidinyl, quinolinyl, isoquinolinyl,quinazolinyl, quinoxalinyl, napthyridinyl, pthalazinyl, azabenzofuryland azaindolyl, each of which may be independently optionallysubstituted with from one to two members selected from the groupconsisting of methyl, -amino, —NHMe, —NMe₂, methoxy, hydroxymethyl andhalogen;D is selected from the group consisting of cyano, S(O)₂R²⁴, halogen,C(O)NR²¹R²², phenyl and heteroaryl; wherein said phenyl or heteroaryl isindependently optionally substituted with one to three same or differenthalogens or from one to three same or different substituents selectedfrom G; wherein heteroaryl is selected from the group consisting ofpyridinyl and oxadiazolyl;A is selected from the group consisting of phenyl, pyridinyl, furyl,thienyl, isoxazolyl and oxazolyl wherein said phenyl, pyridinyl, furyl,thienyl, isoxazolyl and oxazolyl are independently optionallysubstituted with one to three same or different halogens or from one tothree same or different substituents selected from G;G is selected from the group consisting of (C₁₋₆)alkyl, (C₁₋₆)alkenyl,phenyl, hydroxy, methoxy, halogen, —NR²³C(O)—(C₁₋₆)alkyl, —NR²⁴R²⁵,—S(O)₂NR²⁴R²⁵, COOR²⁶ and —CONR²⁴R²⁵; wherein said (C₁₋₆)alkyl isoptionally substituted with hydroxy, dimethylamino or one to three sameor different halogen;R²⁶ is selected from the group consisting of hydrogen and (C₁₋₆)alkyl;R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, are independently selected from the groupconsisting of hydrogen, (C₁₋₆)alkyl and —(CH₂)_(n)NR²⁷R²⁸;n is 0-6; andR²⁷ and R²⁸ are each independently H or methyl.

A more preferred embodiment are compounds above wherein:

X and W are each N;

or compounds above wherein:

X is C; and

W is N.

Another preferred embodiment are compounds as described above wherein:

R¹⁸ is —C(O)-Ph; and

Y is

Another preferred embodiment are compounds as described above wherein:R³ is methoxy or triazolyl; wherein said triazolyl is optionallysubstituted with one substituent selected from G;

R¹⁰-R¹⁷ are each H; and

G is methyl.

Another preferred embodiment are compounds as described above wherein:R¹ is F, and R³ is 1,2,3-triazolyl attached at position N-1.

Another preferred embodiment are compounds as described above wherein:R¹ is OMe, and R³ is 3-methyl-1,2,4-triazolyl attached at position N-1.

Another preferred embodiment are compounds as described above wherein:R¹ and R³ are each methoxy.

Another preferred embodiment are compounds as described above whereinthe salt is sodium, lysine or tromethamine.

Another preferred embodiment of the invention is a pharmaceuticalcomposition which comprises an antiviral effective amount of a compoundof Formula I, including pharmaceutically acceptable salts thereof, andone or more pharmaceutically acceptable carriers, excipients ordiluents.

Another preferred embodiment is the pharmaceutical composition fromabove, useful for treating infection by HIV, which additionallycomprises an antiviral effective amount of an AIDS treatment agentselected from the group consisting of:

-   -   (a) an AIDS antiviral agent;    -   (b) an anti-infective agent;    -   (c) an immunomodulator; and    -   (d) HIV entry inhibitors.

Also encompassed by the embodiments is a method for treating a mammalinfected with the HIV virus comprising administering to said mammal anantiviral effective amount of a compound of Formula I, includingpharmaceutically acceptable salts thereof, and one or morepharmaceutically acceptable carriers, excipients or diluents.

Another embodiment is method described above, comprising administeringto said mammal an antiviral effective amount of a compound of Formula I,including pharmaceutically acceptable salts thereof, in combination withan antiviral effective amount of an AIDS treatment agent selected fromthe group consisting of an AIDS antiviral agent; an anti-infectiveagent; an immunomodulator; and an HIV entry inhibitor.

Another embodiment is the intermediate compounds of Formula II, usefulin making compounds I,

wherein:X is C or N with the proviso that when X is N, R¹ does not exist;W is C or N with the proviso that when W is N, R² does not exist;V is C;R¹ is hydrogen, methoxy or halogen;R² is hydrogen;R³ is methoxy or heteroaryl, each of which may be independentlyoptionally substituted with one substituent selected from G; whereinheteroaryl is triazolyl, pyrazolyl or oxadiazolyl;L and M are independently selected from the group consisting ofhydrogen, C₁-C₆ alkyl, phenyl, benzyl, trialkylsilyl,-2,2,2-trichloroethoxy and 2-trimethylsilylethoxy with the proviso thatnot more than one of L and M can be hydrogen;Y is selected from the group consisting of

R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ are each independently H ormethyl, with the proviso that not more than two of R¹⁰-R¹⁷ are methyl;R¹⁸ is selected from the group consisting of C(O)-phenyl,C(O)-pyridinyl, pyridinyl, pyrimidinyl, quinolinyl, isoquinolinyl,quinazolinyl, quinoxalinyl, napthyridinyl, pthalazinyl, azabenzofuryland azaindolyl, each of which may be independently optionallysubstituted with from one to two members selected from the groupconsisting of methyl, -amino, —NHMe, —NMe₂, methoxy, hydroxymethyl andhalogen;D is selected from the group consisting of cyano, S(O)₂R²⁴, halogen,C(O)NR²¹R²², phenyl and heteroaryl; wherein said phenyl or heteroaryl isindependently optionally substituted with one to three same or differenthalogens or from one to three same or different substituents selectedfrom G; wherein heteroaryl is selected from the group consisting ofpyridinyl and oxadiazolyl;A is selected from the group consisting of phenyl, pyridinyl, furyl,thienyl, isoxazolyl and oxazolyl wherein said phenyl, pyridinyl, furyl,thienyl, isoxazolyl and oxazolyl are independently optionallysubstituted with one to three same or different halogens or from one tothree same or different substituents selected from G;G is selected from the group consisting of (C₁₋₆)alkyl, (C₁₋₆)alkenyl,phenyl, hydroxy, methoxy, halogen, —NR²³C(O)—(C₁₋₆)alkyl, —NR²⁴R²⁵,—S(O)₂NR²⁴R²⁵, COOR²⁶ and —CONR²⁴R²⁵; wherein said (C₁₋₆)alkyl isoptionally substituted with hydroxy, dimethylamino or one to three sameor different halogen;R²⁶ is selected from the group consisting of hydrogen and (C₁₋₆)alkyl;R²⁰, R²¹, R²², R²³, R²⁴, R²⁵ are independently selected from the groupconsisting of hydrogen, (C₁₋₆)alkyl and —(CH₂)_(n)NR²⁷R²⁸;R²⁷ and R²⁸ are each independently H or methyl; andn is 0-6.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates AUC (Area Under the Curve) Versus Dosage in HumanClinical Trials for Compound IVa.

FIG. 2 illustrates AUC for Compound IVa and Prodrug Iab Under Fastingand Fed Conditions in Dog and Human Studies

FIG. 3 illustrates IVc Oral AUC in Rats versus Dose Plots

FIG. 4 illustrates IVc Oral Cmax in Rats versus Dose Plots

FIG. 5 illustrates Plasma Profiles of IVc in Rats After Oral Dosing ofIc

FIG. 6 illustrates Comparison of IVa Cmax and AUC in Male Rats GivenEither IVa or Iab

FIG. 7 illustrates Comparison of IVa Cmax and AUC in Dogs Given EitherIVa or Iab

FIG. 8 illustrates Hydrolysis of tab in Human Placental ALP Solutionsand the Formation of IVa

FIG. 9 illustrates Plasma Concentration Versus Time Profiles of Iab andIVa Following IV and Oral Administration of Iab in Rats and from theHistorical Data of IVa in Rats

FIG. 10 illustrates Plasma Concentration Versus Time Profiles of Iab andIVa Following IV and Oral Administration of Iab in Dogs and from theHistorical Data of IVa in Dogs

FIG. 11 illustrates Plasma Concentration Versus Time Profiles of Iab andIVa Following IV and Oral Administration of Iab in Monkeys and from theHistorical Data of IVa in Monkeys

FIG. 12 illustrates Comparison of IVb Cmax and AUC in Male Rats GivenEither IVb or Ibb

FIG. 13 illustrates Comparison of IVb Cmax and AUC in Dogs Given EitherIVb or Ibb

FIG. 14 illustrates Hydrolysis of Ibb in Human Placental ALP Solutionsand the Formation of IVb

FIG. 15 illustrates Plasma Concentration Versus Time Profiles of Ibb andIVb Following IV and Oral Administration of Ibb in the Rat and theHistorical Data of IVb in the Rat

FIG. 16 illustrates Plasma Concentration Versus Time Profiles of Ibb andIVb Following IV and Oral Administration of Ibb in the Dog and theHistorical Data of IVb in the Dog

FIG. 17 illustrates Plasma Concentration Versus Time Profiles of Ibb andIVb Following IV and Oral Administration of Ibb in the Monkey and theHistorical Data of IVb in the Monkey

FIG. 18 illustrates Comparison of IVc Cmax and AUC in Male Rats GivenEither IVc or Icb

FIG. 19 illustrates Comparison of IVc Cmax and AUC in Dogs Given EitherIVc or Icb

FIG. 20 illustrates Hydrolysis of Icb in Human Placental ALP Solutionsand the Formation of IVc

FIG. 21 illustrates Plasma Concentration Versus Time Profiles of Icb andIVc Following IV and Oral Administration of Icb in the Rat and theHistorical Data of IVc in the Rat

FIG. 22 illustrates Plasma Concentration Versus Time Profiles of Icb andIVc Following IV and Oral Administration of Icb in the Dog and theHistorical Data of IVc in the Dog

FIG. 23 illustrates Plasma Concentration Versus Time Profiles of Icb andIVc Following IV and Oral Administration of Icb in the Monkey and theHistorical Data of IVc in the Monkey

DETAILED DESCRIPTION OF THE INVENTION

Since the compounds of the present invention, may possess asymmetriccenters and therefore occur as mixtures of diastereomers andenantiomers, the present invention includes the individualdiastereoisomeric and enantiomeric forms of the compounds of Formula Iin addition to the mixtures thereof.

DEFINITIONS

The term “C₁₋₆ alkyl” as used herein and in the claims (unless specifiedotherwise) mean straight or branched chain alkyl groups such as methyl,ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, amyl, hexyl and thelike.

“Halogen” refers to chlorine, bromine, iodine or fluorine.

An “aryl” group refers to an all carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, napthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted the substituted group(s) is preferably one or more selectedfrom alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy,alkoxy, aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy,thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halogen,nitro, carbonyl, O-carbamyl, N-carbamyl,

C-amido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamido,trihalomethyl, ureido, amino and —NR^(x)R^(y), wherein R^(x) and R^(y)are independently selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, carbonyl, C-carboxy, sulfonyl, trihalomethyl, and,combined, a five- or six-member heteroalicyclic ring.

As used herein, a “heteroaryl” group refers to a monocyclic or fusedring (i.e., rings which share an adjacent pair of atoms) group having inthe ring(s) one or more atoms selected from the group consisting ofnitrogen, oxygen and sulfur and, in addition, having a completelyconjugated pi-electron system. Unless otherwise indicated, theheteroaryl group may be attached at either a carbon or nitrogen atomwithin the heteroaryl group. It should be noted that the term heteroarylis intended to encompass an N-oxide of the parent heteroaryl if such anN-oxide is chemically feasible as is known in the art. Examples, withoutlimitation, of heteroaryl groups are furyl, thienyl, benzothienyl,thiazolyl, imidazolyl, oxazolyl, oxadiazolyl, thiadiazolyl,benzothiazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl,pyrrolyl, pyranyl, tetrahydropyranyl, pyrazolyl, pyridyl, pyrimidinyl,quinolinyl, isoquinolinyl, purinyl, carbazolyl, benzoxazolyl,benzimidazolyl, indolyl, isoindolyl, pyrazinyl. diazinyl, pyrazine,triazinyltriazine, tetrazinyl, and tetrazolyl. When substituted thesubstituted group(s) is preferably one or more selected from alkyl,cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,heteroaryloxy, heteroalicycloxy, thiohydroxy, thioaryloxy,thioheteroaryloxy, thioheteroalicycloxy, cyano, halogen, nitro,carbonyl, O-carbamyl, N-carbamyl, C-amido, N-amido, C-carboxy,O-carboxy, sulfinyl, sulfonyl, sulfonamido, trihalomethyl, ureido,amino, and —NR^(x)R^(y), wherein R^(x) and R^(y) are as defined above.

As used herein, a “heteroalicyclic” group refers to a monocyclic orfused ring group having in the ring(s) one or more atoms selected fromthe group consisting of nitrogen, oxygen and sulfur. Rings are selectedfrom those which provide stable arrangements of bonds and are notintended to encomplish systems which would not exist. The rings may alsohave one or more double bonds. However, the rings do not have acompletely conjugated pi-electron system. Examples, without limitation,of heteroalicyclic groups are azetidinyl, piperidyl, piperazinyl,imidazolinyl, thiazolidinyl, 3-pyrrolidin-1-yl, morpholinyl,thiomorpholinyl and tetrahydropyranyl. When substituted the substitutedgroup(s) is preferably one or more selected from alkyl, cycloalkyl,aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,heteroaryloxy, heteroalicycloxy, thiohydroxy, thioalkoxy, thioaryloxy,thioheteroaryloxy, thioheteroalicycloxy, cyano, halogen, nitro,carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, C-thioamido, N-amido, C-carboxy, O-carboxy,sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido,trihalomethanesulfonyl, silyl, guanyl, guanidino, ureido, phosphonyl,amino and —NR^(x)R^(y), wherein R^(x) and R^(y) are as defined above.

An “alkyl” group refers to a saturated aliphatic hydrocarbon includingstraight chain and branched chain groups. Preferably, the alkyl grouphas 1 to 20 carbon atoms (whenever a numerical range; e.g., “1-20”, isstated herein, it means that the group, in this case the alkyl group maycontain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to andincluding 20 carbon atoms). More preferably, it is a medium size alkylhaving 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having1 to 4 carbon atoms. The alkyl group may be substituted orunsubstituted. When substituted, the substituent group(s) is preferablyone or more individually selected from trihaloalkyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy,heteroalicycloxy, thiohydroxy, thioalkoxy, thioaryloxy,thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, nitro, carbonyl,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, C-thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl,sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, andcombined, a five- or six-member heteroalicyclic ring.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share and adjacent pair of carbon atoms) groupwherein one or more rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene and adamantane. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is preferably one or more individually selectedfrom alkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,heteroaryloxy, heteroalicycloxy, thiohydroxy, thioalkoxy, thioaryloxy,thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, nitro, carbonyl,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, C-thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl,sulfonamido, trihalo-methanesulfonamido, trihalomethanesulfonyl, silyl,guanyl, guanidino, ureido, phosphonyl, amino and —NR^(x)R^(y) with R^(x)and R^(y) as defined above.

An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbondouble bond.

An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbontriple bond.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl groupas defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

A “heteroaryloxy” group refers to a heteroaryl-O— group with heteroarylas defined herein.

A “heteroalicycloxy” group refers to a heteroalicyclic-O— group withheteroalicyclic as defined herein.

A “thiohydroxy” group refers to an —SH group.

A “thioalkoxy” group refers to both an S-alkyl and an —S-cycloalkylgroup, as defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

A “thioheteroaryloxy” group refers to a heteroaryl-S— group withheteroaryl as defined herein.

A “thioheteroalicycloxy” group refers to a heteroalicyclic-S— group withheteroalicyclic as defined herein.

A “carbonyl” group refers to a —C(═O)—R″ group, where R″ is selectedfrom the group consisting of hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon), as each is definedherein.

An “aldehyde” group refers to a carbonyl group where R″ is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ as definedherein.

A “Keto” group refers to a —CC(═O)C— group wherein the carbon on eitheror both sides of the C═O may be alkyl, cycloalkyl, aryl or a carbon of aheteroaryl or heteroaliacyclic group.

A “trihalomethanecarbonyl” group refers to a Z₃CC(═O)— group with said Zbeing a halogen.

A “C-carboxy” group refers to a —C(═O)O—R″ groups, with R″ as definedherein.

An “O-carboxy” group refers to a R″C(═O)O— group, with R″ as definedherein.

A “carboxylic acid” group refers to a C-carboxy group in which R″ ishydrogen.

A “trihalomethyl” group refers to a —CZ₃, group wherein Z is a halogengroup as defined herein.

A “trihalomethanesulfonyl” group refers to an Z₃CS(═O)₂— groups with Zas defined above.

A “trihalomethanesulfonamido” group refers to a Z₃CS(═O)₂NR^(x)— groupwith Z and R^(X) as defined herein.

A “sulfinyl” group refers to a —S(═O)—R″ group, with R″ as definedherein and, in addition, as a bond only; i.e., —S(O)—.

A “sulfonyl” group refers to a —S(═O)₂R″ group with R″ as defined hereinand, in addition as a bond only; i.e., —S(O)₂—.

A “S-sulfonamido” group refers to a —S(═O)₂NR^(X)R^(Y), with R^(X) andR^(Y) as defined herein.

A “N-Sulfonamido” group refers to a R″S(═O)₂NR_(X)— group with R_(x) asdefined herein.

A “O-carbamyl” group refers to a —OC(═O)NR^(x)R^(y) as defined herein.

A “N-carbamyl” group refers to a R^(x)OC(═O)NR^(y) group, with R^(x) andR^(y) as defined herein.

A “O-thiocarbamyl” group refers to a —OC(═S)NR^(x)R^(y) group with R^(x)and R^(y) as defined herein.

A “N-thiocarbamyl” group refers to a R^(x)OC(═S)NR^(y)— group with R^(x)and R^(y) as defined herein.

An “amino” group refers to an —NH₂ group.

A “C-amido” group refers to a —C(═O)NR^(x)R^(y) group with R^(x) andR^(y) as defined herein.

A “C-thioamido” group refers to a —C(═S)NR^(x)R^(y) group, with R^(x)and R^(y) as defined herein.

A “N-amido” group refers to a R^(x)C(═O)NR^(y)— group, with R^(x) andR^(y) as defined herein.

An “ureido” group refers to a —NR^(x)C(═O)NR^(y)R^(y2) group with R^(x)and R^(y) as defined herein and R^(y2) defined the same as R^(x) andR^(y).

An “thioureido” group refers to a —NR^(x)C(═S)NR^(y)R^(y2) group withR^(x) and R^(y) as defined herein and R^(y2) defined the same as R^(x)and R^(y).

A “guanidino” group refers to a —R^(x)NC(═N)NR^(y)R^(y2) group, withR^(x), R^(y) and R^(y2) as defined herein.

A “guanyl” group refers to a R^(x)R^(y)NC(═N)— group, with R^(x) andR^(Y) as defined herein.

A “cyano” group refers to a —CN group.

A “silyl” group refers to a —Si(R″)₃, with R″ as defined herein.

A “phosphonyl” group refers to a P(═O)(OR^(x))₂ with R^(x) as definedherein.

A “hydrazino” group refers to a —NR^(x)NR^(y)R^(y2) group with R^(x),R^(y) and R^(y2) as defined herein.

Any two adjacent R groups may combine to form an additional aryl,cycloalkyl, heteroaryl or heterocyclic ring fused to the ring initiallybearing those R groups.

It is known in the art that nitrogen atoms in heteroaryl systems can be“participating in a heteroaryl ring double bond”, and this refers to theform of double bonds in the two tautomeric structures which comprisefive-member ring heteroaryl groups. This dictates whether nitrogens canbe substituted as well understood by chemists in the art. The disclosureand claims of the present invention are based on the known generalprinciples of chemical bonding. It is understood that the claims do notencompass structures known to be unstable or not able to exist based onthe literature.

Physiologically acceptable salts of the prodrug compounds disclosedherein are within the scope of this invention. The term“pharmaceutically acceptable salt” as used herein and in the claims isintended to include nontoxic base addition salts. The term“pharmaceutically acceptable salt” as used herein is also intended toinclude salts of acidic groups, such as a carboxylate or phosphate orphosphate mono ester, with such counterions as ammonium, alkali metalsalts, particularly sodium or potassium, alkaline earth metal salts,particularly calcium or magnesium, transition metal salts such as zincand salts with suitable organic bases such as lower alkylamines(methylamine, ethylamine, cyclohexylamine, and the like) or withsubstituted lower alkylamines (e.g. hydroxyl-substituted alkylaminessuch as diethanolamine, triethanolamine or mono tromethamine (alsocalled TRIS or2-amino-2-(hydroxymethyl)propane-1,3-diol)tris(hydroxymethyl)-aminomethane),lysine, arginine, histidine, N-methylglucamine, or with bases such aspiperidine or morpholine. It is understood that both pharmaceuticallyacceptable salts, when isolated in solid or crystalline form, alsoinclude hydrates or water molecules entrapped within the resultingCompound I substance. Stoichiometry possibilities are well known tothose in the art. Discussions of pharmaceutically acceptable salts andlists of possible salts are contained in the following references:

-   Preparation of water-soluble compounds through salt formation.    Stahl, P. Heinrich. Cosmas Consult, Freiburg im Breisgau, Germany.    Editor(s): Wermuth, Camille Georges. Practice of Medicinal Chemistry    (2nd Edition) (2003), 601-615. Publisher: Elsevier, London, UK    CODEN: 69EOEZ.-   Handbook of pharmaceutical salts: properties, selection, and use by    Stahl, P. Heinrich, Wermuth, Camille G., International Union of Pure    and Applied Chemistry. Weinheim; New York: VHCA; Wiley-VCH, 2002.

In another aspect of the invention, novel phosphate ester intermediateCompounds II are disclosed.

In the case of phosphate esters, the possibility of mono or bis existsand both are covered by this invention.

In the method of the present invention, the term “antiviral effectiveamount” means the total amount of each active component of the methodthat is sufficient to show a meaningful patient benefit, i.e., healingof acute conditions characterized by inhibition of the HIV infection.When applied to an individual active ingredient, administered alone, theterm refers to that ingredient alone. When applied to a combination, theterm refers to combined amounts of the active ingredients that result inthe therapeutic effect, whether administered in combination, serially orsimultaneously. The terms “treat, treating, treatment” as used hereinand in the claims means preventing or ameliorating diseases associatedwith HIV infection.

The present invention is also directed to combinations of the compoundswith one or more agents useful in the treatment of AIDS. For example,the compounds of this invention may be effectively administered, whetherat periods of pre-exposure and/or post-exposure, in combination witheffective amounts of the AIDS antivirals, immunomodulators,antiinfectives, or vaccines, such as those in the following table.

Drug Name Manufacturer Indication ANTIVIRALS 097 Hoechst/Bayer HIVinfection, AIDS, ARC (non-nucleoside reverse transcriptase (RT)inhibitor) Amprenavir Glaxo Wellcome HIV infection, 141 W94 AIDS, ARC GW141 (protease inhibitor) Abacavir (1592U89) Glaxo Wellcome HIVinfection, GW 1592 AIDS, ARC (RT inhibitor) Acemannan Carrington LabsARC (Irving, TX) Acyclovir Burroughs Wellcome HIV infection, AIDS, ARC,in combination with AZT AD-439 Tanox Biosystems HIV infection, AIDS, ARCAD-519 Tanox Biosystems HIV infection, AIDS, ARC Adefovir dipivoxilGilead Sciences HIV infection AL-721 Ethigen ARC, PGL (Los Angeles, CA)HIV positive, AIDS Alpha Interferon Glaxo Wellcome Kaposi's sarcoma, HIVin combination w/Retrovir Ansamycin Adria Laboratories ARC LM 427(Dublin, OH) Erbamont (Stamford, CT) Antibody which Advanced BiotherapyAIDS, ARC Neutralizes pH Concepts Labile alpha aberrant (Rockville, MD)Interferon AR177 Aronex Pharm HIV infection, AIDS, ARC Beta-fluoro-ddANat'l Cancer Institute AIDS-associated diseases BMS-232623 Bristol-MyersSquibb/ HIV infection, (CGP-73547) Novartis AIDS, ARC (proteaseinhibitor) BMS-234475 Bristol-Myers Squibb/ HIV infection, (CGP-61755)Novartis AIDS, ARC (protease inhibitor) CI-1012 Warner-Lambert HIV-1infection Cidofovir Gilead Science CMV retinitis, herpes, papillomavirusCurdlan sulfate AJI Pharma USA HIV infection Cytomegalovirus MedImmuneCMV retinitis Immune globin Cytovene Syntex Sight threateningGanciclovir CMV peripheral CMV retinitis Delaviridine Pharmacia-UpjohnHIV infection, AIDS, ARC (RT inhibitor) Dextran Sulfate Ueno Fine Chem.AIDS, ARC, HIV Ind. Ltd. (Osaka, positive Japan) asymptomatic ddCHoffman-La Roche HIV infection, AIDS, Dideoxycytidine ARC ddIBristol-Myers Squibb HIV infection, AIDS, Dideoxyinosine ARC;combination with AZT/d4T DMP-450 AVID HIV infection, (Camden, NJ) AIDS,ARC (protease inhibitor) Efavirenz DuPont Merck HIV infection, (DMP 266)AIDS, ARC (−)6-Chloro-4-(S)- (non-nucleoside RT cyclopropylethynyl-inhibitor) 4(S)-trifluoro- methyl-1,4-dihydro- 2H-3,1-benzoxazin- 2-one,STOCRINE EL10 Elan Corp, PLC HIV infection (Gainesville, GA) FamciclovirSmith Kline herpes zoster, herpes simplex FTC Emory University HIVinfection, AIDS, ARC (reverse transcriptase inhibitor) GS 840 Gilead HIVinfection, AIDS, ARC (reverse transcriptase inhibitor) HBY097 HoechstMarion HIV infection, Roussel AIDS, ARC (non-nucleoside reversetranscriptase inhibitor) Hypericin VIMRx Pharm. HIV infection, AIDS, ARCRecombinant Human Triton Biosciences AIDS, Kaposi's Interferon Beta(Almeda, CA) sarcoma, ARC Interferon alfa-n3 Interferon Sciences ARC,AIDS Indinavir Merck HIV infection, AIDS, ARC, asymptomatic HIVpositive, also in combination with AZT/ddI/ddC ISIS 2922 ISISPharmaceuticals CMV retinitis KNI-272 Nat'l Cancer Institute HIV-assoc.diseases Lamivudine, 3TC Glaxo Wellcome HIV infection, AIDS, ARC(reverse transcriptase inhibitor); also with AZT Lobucavir Bristol-MyersSquibb CMV infection Nelfinavir Agouron HIV infection, PharmaceuticalsAIDS, ARC (protease inhibitor) Nevirapine Boeheringer HIV infection,Ingleheim AIDS, ARC (RT inhibitor) Novapren Novaferon Labs, Inc. HIVinhibitor (Akron, OH) Peptide T Peninsula Labs AIDS Octapeptide(Belmont, CA) Sequence Trisodium Astra Pharm. CMV retinitis, HIVPhosphonoformate Products, Inc. infection, other CMV infectionsPNU-140690 Pharmacia Upjohn HIV infection, AIDS, ARC (proteaseinhibitor) Probucol Vyrex HIV infection, AIDS RBC-CD4 Sheffield Med. HIVinfection, Tech (Houston, TX) AIDS, ARC Ritonavir Abbott HIV infection,AIDS, ARC (protease inhibitor) Saquinavir Hoffmann- HIV infection,LaRoche AIDS, ARC (protease inhibitor) Stavudine; d4T Bristol-MyersSquibb HIV infection, AIDS, Didehydrodeoxy- ARC thymidine ValaciclovirGlaxo Wellcome Genital HSV & CMV infections Virazole Viratek/ICNasymptomatic HIV Ribavirin (Costa Mesa, CA) positive, LAS, ARC VX-478Vertex HIV infection, AIDS, ARC Zalcitabine Hoffmann-LaRoche HIVinfection, AIDS, ARC, with AZT Zidovudine; AZT Glaxo Wellcome HIVinfection, AIDS, ARC, Kaposi's sarcoma, in combination with othertherapies Tenofovir disoproxil, Gilead HIV infection, fumarate saltAIDS, (Viread ®) (reverse transcriptase inhibitor) Emtriva ® Gilead HIVinfection, (Emtricitabine) AIDS, (reverse transcriptase inhibitor)Combivir ® GSK HIV infection, AIDS, (reverse transcriptase inhibitor)Abacavir succinate GSK HIV infection, (or Ziagen ®) AIDS, (reversetranscriptase inhibitor) Reyataz ® Bristol-Myers Squibb HIV infection(or atazanavir) AIDs, protease inhibitor Fuzeon ® Roche/Trimeris HIVinfection (or T-20) AIDs, viral Fusion inhibitor Lexiva ® GSK/Vertex HIVinfection (or Fosamprenavir AIDs, viral protease calcium) inhibitorIMMUNOMODULATORS AS-101 Wyeth-Ayerst AIDS Bropirimine Pharmacia UpjohnAdvanced AIDS Acemannan Carrington Labs, Inc. AIDS, ARC (Irving, TX)CL246,738 American Cyanamid AIDS, Kaposi's Lederle Labs sarcoma FP-21399Fuki ImmunoPharm Blocks HIV fusion with CD4+ cells Gamma InterferonGenentech ARC, in combination w/TNF (tumor necrosis factor) GranulocyteGenetics Institute AIDS Macrophage Colony Sandoz Stimulating FactorGranulocyte Hoechst-Roussel AIDS Macrophage Colony Immunex StimulatingFactor Granulocyte Schering-Plough AIDS, Macrophage Colony combinationStimulating Factor w/AZT HIV Core Particle Rorer Seropositive HIVImmunostimulant IL-2 Cetus AIDS, in combination Interleukin-2 w/AZT IL-2Hoffman-LaRoche AIDS, ARC, HIV, in Interleukin-2 Immunex combinationw/AZT IL-2 Chiron AIDS, increase in Interleukin-2 CD4 cell counts(aldeslukin) Immune Globulin Cutter Biological Pediatric AIDS, inIntravenous (Berkeley, CA) combination w/AZT (human) IMREG-1 Imreg AIDS,Kaposi's (New Orleans, LA) sarcoma, ARC, PGL IMREG-2 Imreg AIDS,Kaposi's (New Orleans, LA) sarcoma, ARC, PGL Imuthiol Diethyl MerieuxInstitute AIDS, ARC Dithio Carbamate Alpha-2 Schering Plough Kaposi'ssarcoma Interferon w/AZT, AIDS Methionine- TNI Pharmaceutical AIDS, ARCEnkephalin (Chicago, IL) MTP-PE Ciba-Geigy Corp. Kaposi's sarcomaMuramyl-Tripeptide Granulocyte Amgen AIDS, in combination ColonyStimulating w/AZT Factor Remune Immune Response Immunotherapeutic Corp.rCD4 Genentech AIDS, ARC Recombinant Soluble Human CD4 rCD4-IgG AIDS,ARC hybrids Recombinant Biogen AIDS, ARC Soluble Human CD4 InterferonHoffman-La Roche Kaposi's sarcoma Alfa 2a AIDS, ARC, in combinationw/AZT SK&F106528 Smith Kline HIV infection Soluble T4 ThymopentinImmunobiology HIV infection Research Institute (Annandale, NJ) TumorNecrosis Genentech ARC, in combination Factor; TNF w/gamma Interferon

ANTI-INFECTIVES Drug Name Manufacturer Indication Clindamycin withPharmacia Upjohn PCP Primaquine Fluconazole Pfizer Cryptococcalmeningitis, candidiasis Pastille Squibb Corp. Prevention of NystatinPastille oral candidiasis Ornidyl Merrell Dow PCP EflornithinePentamidine LyphoMed PCP treatment Isethionate (IM & IV) (Rosemont, IL)Trimethoprim Antibacterial Trimethoprim/sulfa Antibacterial PiritreximBurroughs Wellcome PCP treatment Pentamidine Fisons Corporation PCPprophylaxis Isethionate for Inhalation Spiramycin Rhone-PoulencCryptosporidial diarrhea Intraconazole- Janssen-Pharm. Histoplasmosis;R51211 cryptococcal meningitis Trimetrexate Warner-Lambert PCPDaunorubicin NeXstar, Sequus Kaposi's sarcoma Recombinant Human OrthoPharm. Corp. Severe anemia Erythropoietin assoc. with AZT therapyRecombinant Human Serono AIDS-related Growth Hormone wasting, cachexiaMegestrol Acetate Bristol-Myers Squibb Treatment of anorexia assoc.W/AIDS Testosterone Alza, Smith Kline AIDS-related wasting Total EnteralNorwich Eaton Diarrhea and Nutrition Pharmaceuticals malabsorptionrelated to AIDS

Additionally, the compounds of the invention herein may be used incombination with another class of agents for treating AIDS which arecalled HIV entry inhibitors. Examples of such HIV entry inhibitors arediscussed in DRUGS OF THE FUTURE 1999, 24(12), pp. 1355-1362; CELL, Vol.9, pp. 243-246, Oct. 29, 1999; and DRUG DISCOVERY TODAY, Vol. 5, No. 5,May 2000, pp. 183-194 and Inhibitors of the entry of HIV into hostcells. Meanwell, Nicholas A.; Kadow, John F. Current Opinion in DrugDiscovery & Development (2003), 6(4), 451-461. Specifically thecompounds can be utilized in combination with other attachmentinhibitors, fusion inhibitors, and chemokine receptor antagonists aimedat either the CCR5 or CXCR4 coreceptor.

It will be understood that the scope of combinations of the compounds ofthis invention with AIDS antivirals, immunomodulators, anti-infectives,HIV entry inhibitors or vaccines is not limited to the list in the aboveTable but includes, in principle, any combination with anypharmaceutical composition useful for the treatment of AIDS.

Preferred combinations are simultaneous or alternating treatments with acompound of the present invention and an inhibitor of HIV proteaseand/or a non-nucleoside inhibitor of HIV reverse transcriptase. Anoptional fourth component in the combination is a nucleoside inhibitorof HIV reverse transcriptase, such as AZT, 3TC, ddC or ddI. A preferredinhibitor of HIV protease is Reyataz® (active ingredient Atazanavir).Typically a dose of 300 to 600 mg is administered once a day. This maybe co-administered with a low dose of Ritonavir (50 to 500 mgs). Anotherpreferred inhibitor of HIV protease is Kaletra®. Another usefulinhibitor of HIV protease is indinavir, which is the sulfate salt ofN-(2(R)-hydroxy-1-(S)-indanyl)-2(R)-phenylmethyl-4-(S)-hydroxy-5-(1-(4-(3-pyridyl-methyl)-2(S)—N′-(t-butylcarboxamido)-piperazinyl))-pentaneamideethanolate, and is synthesized according to U.S. Pat. No. 5,413,999.Indinavir is generally administered at a dosage of 800 mg three times aday. Other preferred protease inhibitors are nelfinavir and ritonavir.Another preferred inhibitor of HIV protease is saquinavir which isadministered in a dosage of 600 or 1200 mg tid. Preferred non-nucleosideinhibitors of HIV reverse transcriptase include efavirenz. Thepreparation of ddC, ddI and AZT are also described in EPO 0,484,071.These combinations may have unexpected effects on limiting the spreadand degree of infection of HIV. Preferred combinations include thosewith the following (1) indinavir with efavirenz, and, optionally, AZTand/or 3TC and/or ddI and/or ddC; (2) indinavir, and any of AZT and/orddI and/or ddC and/or 3TC, in particular, indinavir and AZT and 3TC; (3)stavudine and 3TC and/or zidovudine; (4) zidovudine and lamivudine and141W94 and 1592U89; (5) zidovudine and lamivudine.

In such combinations the compound of the present invention and otheractive agents may be administered separately or in conjunction. Inaddition, the administration of one element may be prior to, concurrentto, or subsequent to the administration of other agent(s).

ABBREVIATIONS

The following abbreviations, most of which are conventionalabbreviations well known to those skilled in the art, are usedthroughout the description of the invention and the examples. Some ofthe abbreviations used are as follows:

-   -   h=hour(s)    -   r.t.=room temperature    -   mol=mole(s)    -   mmol=millimole(s)    -   g=gram(s)    -   mg=milligram(s)    -   mL=milliliter(s)    -   TFA=Trifluoroacetic Acid    -   DCE=1,2-Dichloroethane    -   CH₂Cl₂=Dichloromethane    -   TPAP=tetrapropylammonium perruthenate    -   THF=Tetrahydrofuran    -   DEPBT=3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one    -   DMAP=4-dimethylaminopyridine    -   P-EDC=Polymer supported        1-(3-dimethylaminopropyl)-3-ethylcarbodiimide    -   EDC=1-(3-dimethylaminopropyl)-3-ethylcarbodiimide    -   DMF=N,N-dimethylformamide    -   Hunig's Base=N,N-Diisopropylethylamine    -   MCPBA=meta-Chloroperbenzoic Acid    -   azaindole=1H-Pyrrolo-pyridine    -   4-azaindole=1H-pyaolo[3,2-b]pyridine    -   5-azaindole=1H-Pyrrolo[3,2-c]pyridine    -   6-azaindole=1H-pyrrolo[2,3-c]pyridine    -   7-azaindole=1H-Pyrrolo[2,3-b]pyridine    -   4,6-diazaindole=5H-Pyrrolo[3,2-d]pyrimidine    -   5,6-diazaindole=1H-Pyrrolo[2,3-d]pyridazine    -   5,7-diazaindole=7H-Pyrrolo[2,3-d]pyrimidine    -   PMB=4-Methoxybenzyl    -   DDQ=2,3-Dichloro-5,6-dicyano-1,4-benzoquinone    -   OTf=Trifluoromethanesulfonoxy    -   NMM=4-Methylmorpholine    -   PIP-COPh=1-Benzoylpiperazine    -   NaHMDS=Sodium hexamethyldisilazide    -   EDAC=1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide    -   TMS=Trimethylsilyl    -   DCM=Dichloromethane    -   DCE=Dichloroethane    -   MeOH=Methanol    -   THF=Tetrahydrofuran    -   EtOAc=Ethyl Acetate    -   LDA=Lithium diisopropylamide    -   TMP-Li=2,2,6,6-tetramethylpiperidinyl lithium    -   DME=Dimethoxyethane    -   DIBALH=Diisobutylaluminum hydride    -   HOBT=1-hydroxybenzotriazole    -   CBZ=Benzyloxycarbonyl    -   PCC=Pyridinium chlorochromate    -   TRIS=Tromethamine or 2-amino-2-(hydroxymethyl)propane-1,3-diol        Chemistry

The present invention comprises compounds of Formula I, theirpharmaceutical formulations, and their use in patients suffering from orsusceptible to HIV infection.

Scheme A depicts an overview of the process for preparing the prodrugs Iof the invention from the parent molecules IV.

To elaborate on the method, as shown in Scheme A, the antiviral parentcompound of interest, IV, is converted into the phosphate intermediateII, by N-alkylation with chloride intermediate III, in the presence of asuitable base such as sodium hydride, potassium hydride, sodium amide,sodium t-butoxide, sodium (bis trimethylsilyl)amide, potassium (bistrimethyl silyl)amide, or combinations thereof such as sodium hydrideplus sodium bis(trimethylsilyl)amide. The preparation of reagent III andthe methodology for use in preparing prodrugs by the alkylation hydroxygroups has been described in Y. Ueda et. al. U.S. Pat. No. 6,362,172B2which is incorporated by reference in its entirety. The alkylationconditions, protecting groups, protecting group removal, and conditionsfor salt formation are in general applicable to our application despitethe fact that we are alkylating an azaindole in the indole ring ratherthan a hydroxy group. In the current application, from 1.1 to 5.0equivalents of base may be utilized with between 2 and 4 equivalentsbeing preferred. From 1.1 up to 12 equivalents of reagent III may beused with 5 to 10 being preferred depending on the substrate. Thereagent may be added in one portion or incrementally in several portionsover time. A source of iodide ion is usually added to the reaction toprovide increased yields. Elemental iodine is currently preferred as thesource of iodide. 0.1 to 1.5 equivalents of iodine are usually added perazaindole/indole NH being alkylated with 1.0 to 1.2 equivalents ofiodine being preferred since yields are highest. Alternate sources ofiodide include, for example, sodium iodide, lithium iodide, cesiumiodide, copper iodide, or tetrabutyl ammonium iodide. The function ofiodine is presumably to generate the corresponding iodomethyl reagentIIIa in situ from the chloro methyl reagent III. The iodo or bromoreagents corresponding to III could likely be used directly in thereaction in place of the chloride III. The alkylation reaction of step Ais usually carried out in an inert organic solvent such astetrahydrofuran at a temperature from about 0° C. to 50° C., morepreferably between 20° and 40° C. Other anhydrous organic solvents suchas methyl tetrahydrofuran, methyl t-butyl ether, dioxane, ethyleneglycol dimethyl ether, dimethyl acetamide, or N,N-dimethylformamidecould also find utility. Ester intermediate II is then subjected to aconventional deprotection step to remove the protecting groups Pr. Thereagents used in such step will depend on the protecting group used, butwill be well known to those skilled in the art. The most preferredprotecting group is the t-butyl group which can be removed withtrifluoroacetic acid, hydrochloric acid, or formic acid in anappropriate inert organic solvent. The inert solvent can bedichloromethane, or possibly, for example, dichloroethane, toluene, ortrifluoromethyl benzene. In methylene chloride, trifluoroacetic aciddeprotection may be effected using from 1 to 15 equivalents of acid (orthe acid can be measured differently as for example a 5% solution insolvent by volume) and temperatures of between 0° and 40°. In general,the greater excess of TFA employed, the lower the temperature utilized.Exact conditions vary with substrate. Step 3 describes the isolation ofthe free acid or salts which can be formed via many standard ways whichare well known in the art. Generally, following TFA deprotection, anaqueous workup is employed in which the excess acid is neutralized witha base and the organic impurities removed via extraction with an organicsolvent such as ethyl acetate or dichloromethane. For example excessaqueous NaOH may be used to basify the reaction mixture. This is wellknown to any chemist skilled in the art. Reacidification of the aqueousphase to pH 2.5 with aqueous 1N HCl and then extraction with an organicsolvent will provide, after removal of solvent in vacuo, the free acid.The free acid may be converted to inorganic salts by the addition ofappropriate bases in solvents such as water, methanol, ethanol, etc. Forexample, addition of sodium carbonate to an aqueous solution ofphosphate prodrug and adjustment of the pH to approximately 7.6 providesa solution which upon removal of water via lypohilization leaves thedisodium salt of the prodrug. Potassium carbonate could be usedsimilarly. Aqueous solutions of sodium bicarbonate or potassiumbicarbonate could be used similarly. Mono potassium or mono sodium saltscould be generated via careful titration of phosphate acid solutionswith potassium or sodium 2-ethyl hexanoate. Amine salts can be generatedby dissolving the free acid in organic solvents such as ethyl acetate oracetonitrile or low molecular weight alcohols or mixtures of thesesolvents optionally containing water. Some amines potentially useful forsalt formation include: lower alkylamines (methylamine, ethylamine,cyclohexylamine, and the like) or substituted lower alkylamines (e.g.hydroxyl-substituted alkylamines such as diethanolamine, triethanolamineor tris(hydroxymethyl)-aminomethane), lysine, arginine, histidine,N-methylglucamine, or bases such as piperidine or morpholine. Slowaddition of an amine to a stirring solution at low temperature canprovide either the mono of bis amine salt depending on stoichiometry.Amine salts can also be obtained by stirring the solution and removingthe solvent in vacuo rather than be crystallization or precipitation.Recrystallization procedures will vary by compound and salt but areavailable to one skilled in the art. Scheme B depicts a preferredsequence and set of reagents and conditions for carrying out the generalsequence shown in Scheme A. An alternate and in many cases the preferredmethod for carrying out the sequence in step C which includes thedeprotection of the diester to provide the intermediate acid insitufollowed by salt formation in the reaction medium may be utilized. Forexample, heating the diester II in a mixture of water and a watermiscible cosolvent such as for example acetone, methanol, ethanol, orisopropanol can produce the free acid of I in the reaction medium(insitu). A preferred solvent is acetone and isopropanol. Temperaturesbetween ambient and the boiling points of the solvents could beutilized. Typically 40° to 60° C. is in the preferred range. Addition ofa base or more preferably an amine as described above to the reactionmixture containing the free acid in the water and cosolvent can producethe salt directly. If appropriate conditions are selected, the salt, maycrystallize or precipitate directly from the reaction medium and beisolated by filtration and drying. Specific examples are contained inthe experimental section.

The preferred method of preparation of intermediates IIa, IIb, and IIcand of the acids Iac, Ibc and Ic offers a number of significantadvantages over the procedures used initially for preparation of IIa,IIb, and IIc during exploratory research efforts. The initial discoveryroutes for the preparation of all three intermediates II used apreparation of di-tertbutyl chloromethyl phosphate that required the useof relatively expensive tetrabutylammonium di tert-butyl phosphate whichwas reacted with a 10 fold excess of expensive and potentially hazardouschoro iodomethane. The excess volatiles and iodomethane were removed invacuo to provide the crude reagent which was used without furtherpurification. At least a 5 fold excess of this reagent was used to reactwith compounds IV meaning that at least 5 equivalents oftetrabutylammonium phosphate and 50 equivalents of choloriodomethanewere used as compared to quantities of either IVa, b or c. In addition,alkylation of compounds IV with this reagent was achieved using NaH asbase and iodine as an additive to promote alkylation. These conditionsproduced a reaction mixture containing the desired compound II as amajor product and side products which were removed using silica gelchromatography, a tedious, time consuming, and expensive operationespecially on reactions of increasing scale. Failure to remove sideproducts, resulted in products I from the next step, the removal of theprotecting groups, that contained impurities which were very difficultto remove in a satisfactory manner in either a reasonable yield or timeframe.

The improved preparation of di-tertbutyl chloromethyl phosphate utilizesless expensive ditertbutyl potassium phosphate and only an approximate 2fold excess of this reagent as compared to the other reactantchloromethyl sulfonyl chloride. The di-tertbutyl chloromethyl phosphateprepared by this method is isolated in pure form via convenientdistillation.

For the conversion of IVa to IIa, only 1.2 equivalents of this reagentwas used to alkylate compounds IV. In addition, a less reactive andeconomical base, potassium carbonate, was used in conjunction with DMSOto achieve an alkylation in which the compounds II produced aresufficiently free of side products that they can be used withoutchromatographic purification as inputs for the deprotection reaction.The free acid Iac or salts such as Iab for example can be obtained inpure form from Ha prepared in this manner without chromatography.

For the synthesis of compounds IIb and IIc which are slower to reactthan IVa, 2 to 2.5 equivalents of di-terbutyl chloromethyl phosphatewere employed and modified conditions (cesium carbonate as base with KIin the solvent, NMP) were used to alkylate either IVb and IVc andrealize high conversion to IIb and IIc respectively.

Again the new conditions provided compounds II in sufficient purity toallow them to be used to produce compounds I without the need forchromatographic purification. Thus the new conditions reduce thequantities and stoichiometry of reagents needed to prepare the compoundI of this invention and avoids the need for chromatographic purificationof intermediates II. The starting materials employed to prepare thedi-tertbutyl chloromethyl phosphate are more economical and lesshazardous and the final product is produced in higher purity. Finally,the conditions developed for alkylating IVa, IVb, and IVc eliminate theneed for the reactive, flammable sodium hydride base that had been usedin excess and employ either potassium or cesium carbonate.

In the alkylation step, a suitable base can be used such as M₂CO₃ (M islithium, sodium, potassium, rubidium or cesium). For converting IVa toIIa, K₂CO₃ is preferred (1-5 molar equivalents, preferably 2 molarequivalents per mole of IVa). For converting IVb to IIb or IVc to IIc,cesium carbonate is preferred.

Also, a suitable solvent is needed such as dimethylsulfoxide,N-dimethylformamide, acetone, acetonitrile, N-methylpyrrolidinone,formamide, tetrahydrofuran, etc. (2-50 ml/gram of IV, with 5 ml/grampreferred); dimethylsulfoxide is preferred in converting IVa to IIa;N-methylpyrrolidinone is preferred in converting IVb to IIb or IVc toIIc.

A suitable solvent iodine source includes, but is not limited to, MI (Mis, for example, lithium, sodium, potassium, iodine, tetrabutylammonium,etc.); with potassium iodide preferred (0.1-5 molar equivalents/mole ofCompound IV; 2 equivalents preferred).

The alkylating agent, di-tert-butyl chloromethyl phosphate, can be usedin 1-10 molar equivalents per mole of IV; but about 1.2 molarequivalents is preferred.

The reaction temperature can be 10-60° C. (30° C. preferred).

In the deprotecting step, when the two tert-butyl groups are removedfrom IIa to form Iac, this is carried out in the presence of a suitablesolvent such as dichloromethane (preferred), dichloroethane, chloroform,carbontetrachloride, toluene, benzene, etc. (2-50 ml/gram of IV,preferably 10 ml/gram)

For deprotecting IIb and IIc to obtain Ibc and Ic, respectively, this isaccomplished in acetone/water at a temperature of about 40° C.

Additionally, during deprotection of IIa, it is preferred to have anacid present such as trifluoroacetic acid (preferred), hydrochloric,sulfuric, nitric, etc. (2-100 molar equivalents based on IVa, with 15molar equivalents preferred).

Chemistry

General:

Additional preparations of starting materials and precursors arecontained in Wang et. al. U.S. Pat. No. 6,476,034 granted Nov. 5, 2002which is incorporated by reference in its entirety.

All Liquid Chromatography (LC) data were recorded on a Shimadzu LC-10ASliquid chromatograph using a SPD-10AV UV-Vis detector with MassSpectrometry (MS) data determined using a Micromass Platform for LC inelectrospray mode.

LC/MS Method (i.e., Compound Identification)

Column A: YMC ODS-A S7 3.0×50 mm column

Column B: PHX-LUNA C18 4.6×30 mm column

Column C: XTERRA ms C18 4.6×30 mm column

Column D: YMC ODS-A C18 4.6×30 mm column

Column E: YMC ODS-A C18 4.6×33 mm column

Column F: YMC C18 S5 4.6×50 mm column

Column G: XTERRA C18 S7 3.0×50 mm column

Column H: YMC C18 S5 4.6×33 mm column

Column I: YMC ODS-A C18 S7 3.0×50 mm column

Column J: XTERRA C-18 S5 4.6×50 mm column

Column K: YMC ODS-A C18 4.6×33 mm column

Column L: Xterra MS C18 5 uM 4.6×30 mm column

Column M: YMC ODS-A C18 S3 4.6×33 mm column

Standard LC Run Conditions (Used Unless Otherwise Noted):

-   Gradient: 100% Solvent A/0% Solvent B to 0% Solvent A/100% Solvent B-   Solvent A=10% MeOH−90% H₂O−0.1% TFA, Solvent B=90% MeOH−10% H₂O−0.1%    TFA; and R_(t) in min.-   Gradient time: 2 minutes-   Hold time: 1 minute-   Flow rate: 5 mL/min-   Detector Wavelength: 220 nm-   Solvent A: 10% MeOH/90% H₂O/0.1% Trifluoroacetic Acid-   Solvent B: 10% H₂O/90% MeOH/0.1% Trifluoroacetic Acid    Alternate LC Run Conditions B:-   Gradient: 100% Solvent A/0% Solvent B to 0% Solvent A/100% Solvent B-   Solvent A=10% MeOH−90% H₂O−0.1% TFA, Solvent B=90% MeOH−10% H₂O−0.1%    TFA; and R_(t) in min.-   Gradient time: 4 minutes-   Hold time 1 minute-   Flow rate: 4 mL/min-   Detector Wavelength: 220 nm-   Solvent A: 10% MeOH/90% H₂O/0.1% Trifluoroacetic Acid-   Solvent B: 10% H₂O/90% MeOH/0.1% Trifluoroacetic Acid

Compounds purified by preparative HPLC were diluted in MeOH (1.2 mL) andpurified using the following methods on a Shimadzu LC-10A automatedpreparative HPLC system or on a Shimadzu LC-8A automated preparativeHPLC system with detector (SPD-10AV UV-VIS) wavelength and solventsystems (A and B) the same as above.

Preparative HPLC Method (i.e., Compound Purification)

Purification Method: Initial gradient (40% B, 60% A) ramp to finalgradient (100% B, 0% A) over 20 minutes, hold for 3 minutes (100% B, 0%A)

Solvent A: 10% MeOH/90% H₂O/0.1% Trifluoroacetic Acid

Solvent B: 10% H₂O/90% MeOH/0.1% Trifluoroacetic Acid

Column: YMC C18 S5 20×100 mm column

Detector Wavelength: 220 nm

For the experimental procedures below the following HPLC conditions ormodifications from the standard procedures were employed:

HPLC Conditions for Routine LC Purity:

Detection at 254 nm; Gradient 0-100% B/A; A 10% CH3CN—90% H2O—0.1% TFA,B 90% CH3CN—10% H2O—0.1% TFA; Gradient time 4 min; Column YMC ODS-AQ orORD-A 4.6×50 mm 3 micron.

HPLC Conditions for LC/MS Analysis:

Column J: XTERRA C-18 S5 4.6×50 mm column, Gradient: 100% Solvent A/0%Solvent B to 0% Solvent A/100% Solvent B

Solvent A=10% MeOH−90% H2O−0.1% TFA, Solvent B=90% MeOH−10% H2O−0.1%TFA; and Rt in min; Gradient time: 3 minutes; Flow rate: 4 mL/min;Detector Wavelength: 220 nm

Starting materials, can be purchased from commercial sources or preparedusing literature procedures.

Preparation of Parent Compounds IV:

The preparation of parent Compounds IV has been described previously inthe following, all of which are herein incorporated by reference intheir entirety as follows:

-   U.S. Pat. No. 6,469,006 granted Oct. 22, 2002 to W. S. Blair et al;-   U.S. Pat. No. 6,476,034 granted Nov. 5, 2002 to Wang et al;-   U.S. Pat. No. 6,573,262 granted Jun. 3, 2003 to Meanwell et al;-   U.S. Ser. No. 10/630,278 filed Jul. 30, 2003 to J. Kadow et al;    which is a continuation-in-part of U.S. Ser. No. 10/214,982 filed    Aug. 7, 2002, which is a continuation-in-part of U.S. Ser. No.    10/038,306 filed Jan. 2, 2002, which corresponds to PCT WO    02/062423, filed Jan. 2, 2002, published Aug. 15, 2002;-   U.S. Ser. No. 10/871,931 filed Jun. 18, 2004 to Yeung et al;-   U.S. Ser. No. 10/762,108 filed Jan. 21, 2004 to Wang et al,    corresponding to PCT WO 2004/043337 published May 27, 2004.

Select detailed procedures are provided below:

Typical Procedure for the Preparation of Intermediates for thePreparation of Parent Compounds IV

1) Preparation of Azaindole 1

Preparation of azaindole, Method A: Preparation of 7-Chloro-6-azaindole1e: 2-Chloro-3-nitropyridine 22e (5.0 g) was dissolved in dry THF (200ml). After the solution was cooled down to −78° C., an excess of vinylmagnesium bromide (1.0 M in THF, 100 ml) was added. Then, the reactionwas left at −20° C. for eight hours before being quenched with 20% NH₄Cl(150 ml). The aqueous phase was extracted with EtOAc (3×150 ml). Thecombined organic layer was dried over MgSO₄. After filtration andconcentration, the crude product was purified by silica gel columnchromatography to afford 1.5 g of 7-chloro-6-azaindole 1e in 31% yield.

Compounds 5an, IVa and 5ap are described below.

Compound 1am, 4-bromo-7-chloro-6-azaindole (yellow solid) was preparedby the same method used for azaindole 1e but the starting materialemployed was 5-bromo-2-chloro-3-nitropyridine. (available from Aldrich,Co.). MS m/z: (M+H)⁺ calcd for C₇H₅BrClN₂: 230.93; found 231.15. HPLCretention time: 1.62 minutes (column B).

Compound 1an (4-methoxy-7-chloro-6-azaindole) and compound 1ao(4,7-dimethoxy-6-azaindole): A mixture of 4-bromo-7-chloro-6-azaindole(1 g), CuI (0.65 g) and NaOMe (4 ml, 25%) in MeOH (16 ml) was heated at110-120° C. for 16 hours in a sealed tube. After cooling to ambienttemperature, the reaction mixture was neutralized with 1N HCl to achievepH 7. The aqueous solution was extracted with EtOAc (3×30 ml). Then thecombined organic layer was dried over MgSO₄ and concentrated in vacuo toafford a residue, which was purified by silica gel (50 g) chromatographyusing 1:7 EtOAc:hexane as the eluent. (Column dimension: 20 mm×30 cm) togive 0.3 g of 4-methoxy-7-chloro-6-azaindole (white solid) and 0.1 g of4,7-dimethoxy-6-azaindole (white solid).

Compound 1an (4-methoxy-7-chloro-6-azaindole). MS m/z: (M+H)⁺ calcd forC₈H₈ClN₂O: 183.03; found 183.09. HPLC retention time: 1.02 minutes(column B).

Compound 1ao (4,7-dimethoxy-6-azaindole). ¹H NMR (500 MHz, CDCl₃) δ 7.28(m, 2H), 6.63 (m, 1H), 4.14 (s, 3H), 3.95 (s, 3H). MS m/z: (M+H)⁺ calcdfor C₉H₁₁N₂O₂: 179.08; found 179.05. HPLC retention time: 1.36 minutes(column B).

Acylation of Azaindole, Method B: Preparation of Methyl(5-azaindol-3-yl)-oxoacetate 2b

5-Azaindole (1b) (0.5 g, 4.2 mmol) was added to a suspension of AlCl₃(2.8 g, 21.0 mmol) in CH₂Cl₂ (100 ml). Stirring was continued at roomtemperature for 1 hour before methyl chlorooxoacetate (2.5 g, 21.0 mmol)was added dropwise. The reaction was stirred for 8 hours. After 20 ml ofMeOH was added cautiously to quench the reaction, solvents were removedunder vacuum. The solid residue was purified by silica gel columnchromatography (EtOAc/MeOH=10:1) to afford 0.6 g (70%) of the acylatedproduct 2b.

Characterization of Compounds 2:

Compound 2b (Methyl (5-azaindol-3-yl)-oxoacetate)

¹H NMR (500 MHz, CD₃OD) δ 9.61 (s, 1H), 9.02 (s, 1H), 8.59 (d, 1H,J=6.63 Hz), 8.15 (d, 1H, J=6.60 Hz), 4.00 (s, 3H); ¹³C NMR (125 MHz,CD₃OD) δ 178.9, 163.0, 145.6, 144.2, 138.3, 135.0, 124.7, 116.3, 112.1,53.8. MS m/z: (M+H)⁺ calcd for C₁₀H₉N₂O₃: 205.06; found 205.04. HPLCretention time: 0.32 minutes (column A).

Compound 2ao (Ethyl (4,7-dimethoxy-6-azaindol-3-yl)-oxoacetate) wasprepared by the same method as used for compound 2b but the startingmaterial employed was 4,7-dimethoxy-6-azaindole. The compound waspurified by silica gel chromatography using 2:3 EtOAc:Hexane as theeluent to give a yellow oil: ¹H NMR (500 MHz, CDCl₃) δ 9.50 (s, 1H),8.21 (s, 1H), 7.47 (s, 1H), 4.39 (q, 2H, d=7.05 Hz), 4.13 (s, 3H), 3.93(s, 3H), 1.40 (t, 3H, d=7.2 Hz). MS m/z: (M+H)⁺ calcd for C₁₃H₁₅N₂O₅:279.10; found 279.16. HPLC retention time: 1.28 minutes (column B).

2) Preparation of Potassium Azaindole 3-glyoxylate 3

Preparation of Potassium (7-azaindol-3-yl)-oxoacetate 3a

Compound 2a (43 g, 0.21 mol) and K₂CO₃ (56.9 g, 0.41 mol) were dissolvedin MeOH (200 ml) and H₂O (200 ml). After 8 hours, product 3aprecipitated out from the solution. Filtration afforded 43 g of compound3a as a white solid in 90.4% yield.

Characterization of Compounds 3:

Compound 3a, Potassium (7-azaindol-3-yl)-oxoacetate

¹H NMR (300 MHz, DMSO-d₆) δ 8.42 (d, 1H, J=7.86 Hz), 8.26 (d, 1H, J=4.71Hz), 8.14 (s, 1H), 7.18 (dd, 1H, J=7.86, 4.71 Hz); ¹³C NMR (75 MHz,DMSO-d₆) δ 169.4, 148.9, 143.6, 135.1, 129.3, 118.2, 117.5, 112.9. MSm/z: (M+H)⁺ of the corresponding acid of compound 3a (3a−K+H) calcd forC₉H₇N₂O₃: 191.05; found 190.97. HPLC retention time: 0.48 minutes(column A).

Compound 3ao (Potassium (4,7-dimethoxy-6-azaindol-3-yl)-oxoacetate) wasprepared (as a yellow solid), by the same method used to preparecompound 3a except Ethyl (4,7-dimethoxy-6-azaindol-3-yl)-oxoacetate wasemployed as the starting material. MS m/z: (M+H)⁺ of the correspondingacid of compound 3ao (M−K+H)⁺ calcd for C₁₁H₁₁N₂O₅: 251.07; found251.09. HPLC retention time: 0.69 minutes (column B).

Example Procedure Prep of 5a

Preparation of(R)—N-(benzoyl)-3-methyl-N′-[(7-azaindol-3-yl)-oxoacetyl]-piperazine 5a

Potassium 7-azaindole 3-glyoxylate 3a (25.4 g, 0.111 mol),(R)-3-methyl-N-benzoylpiperazine 4a (22.7 g, 0.111 mol),3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) (33.3 g,0.111 mol) and Hunig's Base (28.6 g, 0.222 mol) were combined in 500 mlof DMF. The mixture was stirred at room temperature for 8 hours.

DMF was removed via evaporation at reduced pressure and the residue waspartitioned between ethyl acetate (2000 ml) and 5% Na₂CO₃ aqueoussolution (2×400 ml). The aqueous layer was extracted with ethyl acetate(3×300 ml). The organic phase combined and dried over anhydrous MgSO₄.Concentration in vacuo provided a crude product, which was purified bysilica gel column chromatography with EtOAc/MeOH (50:1) to give 33 g ofproduct 5a in 81% yield.

Characterization of Compounds 5 with the Following Sub-Structure:

Compound 5a, n=2, R₇₋₁₃═H, R₁₄═(R)-Me,(R)—N-(benzoyl)-3-methyl-N′-[(7-azaindol-3-yl)-oxoacetyl]-piperazine

¹H NMR (300 MHz, CD₃OD) δ 8.57 (d, 1H, J=5.97 Hz), 8.38 (d, 1H, J=4.20Hz), 8.27 (m, 1H), 7.47 (s, 5H), 7.35 (t, 1H, J=5.13 Hz), 4.75-2.87 (m,7H), 1.31 (b, 3H); ¹³C NMR (75 MHz, CD₃OD) δ 185.6, 172.0, 166.3, 148.9,144.6, 137.0, 134.8, 130.2, 129.9, 128.4, 126.6, 118.6, 118.0, 112.2,61.3, 50.3, 45.1, 35.5, 14.9, 13.7. MS m/z: (M+H)⁺ calcd for C₂₁H₂₁N₄O₃:377.16; found 377.18. HPLC retention time: 1.21 minutes (column A).Anal. Calcd for C₂₁H₂₀N₄O₃: C, 67.01; H, 5.36; N, 14.88. Found: C,66.01; H, 5.35; N, 14.61.

Compound IVa,N-(benzoyl)-N′-[(4,7-dimethoxy-6-azaindol-3-yl)-oxoacetyl]piperazine,was prepared by the same method used to prepare compound 5a but thestarting material was Potassium(4,7-dimethoxy-6-azaindol-3-yl)-oxoacetate. The compound was purified bysilica gel chromatography using EtOAc as the eluting solvent to give awhite solid. ¹H NMR (500 MHz, DMSO-d₆) δ 13.0 (s, 1H), 8.15 (s, 1H),7.40 (m, 6H), 4.00 (s, 3H), 3.83 (s, 3H), 3.63-3.34 (m, 8H); ¹³C NMR(125 MHz, DMSO-d₆) δ 185.5, 169.3, 166.5, 146.2, 145.7, 136.6, 135.3,129.6, 128.4, 126.9, 122.2, 122.1, 119.2, 114.4, 56.8, 52.9, 45.5, 39.9.MS m/z: (M+H)⁺ calcd for C₂₂H₂₃N₄O₅: 423.17; found 423.19. HPLCretention time: 1.33 minutes (column B). Anal. Calcd. For C₂₂H₂₁N₄O₅: C,62.7; H, 5.02; N, 13.29. Found: C, 61.92; H, 5.41; 13.01. Melting Point:229.5-232° C.

Procedures for Preparation of Parent Compound IVc Preparation of3-methyl-1,2,4-triazole (2-81)

Procedure:

A solid mixture of formic hydrazide (68 g, 1.13 mol) and thioacetamide(85 g, 1.13 mol) in a 500 mL-round bottom flask was heated with stirringat 150° C. (oil bath temp.) for 1.5 hrs with a gentle stream ofnitrogen, removing H₂S and water (about 18 mL of liquid collected)formed during the reaction. The reaction mixture was distilled underreduced pressure, collecting 60.3 g (0.726 mol, Y. 63.3%) of the titlecompound at 102° C./0.35-1 mmHg as a white solid after removing a liquidforerun: ¹H NMR (CDCl₃) δ ppm 2.51 (3H, s, 3-Me), 8.03 (1H, s, 5-H), 9.5(1H, br, NH); TLC Rf (10% MeOH/CH₂Cl₂)=0.3 (phosphomolybdate-charring,white spot). Reference: Vanek, T.; Velkova, V.; Gut, Jiri Coll. Czech.Chem. Comm. 1985, 49, 2492.

Preparation of 3-81

Procedure:

A 500 mL round bottom flask was loaded with4-methoxy-7-chloro-6-azaindole 2e (9.1 g, 50 mmol; dried in vacuo),potassium carbonate (13.8 g, 100 mmol, 2 eq.), copper powder (6.35 g,100 mmol, 2 eq.), and 3-methyl-1,2,4-triazole (83 g, 1.0 mol, 20 eq.).The solid mixture was heated to melt at 170-175° C. (external oil bathtemperature) under a gentle stream of anhydrous nitrogen for 12 h, bywhich time HPLC analysis indicated that the amount of the peak for thestarting material had become 5-30% and the desired product peak becomesabout 45% with isomeric by-product peak becomes 15%. As the reactionmixture cooled, MeOH (150 mL) was added slowly to the warm, stirredmixture. Upon cooling, the insoluble material (copper powder) wasfiltered through a Celite pad, and rinsed with methanol. The filtratewas concentrated in vacuo to a thick paste which was diluted with water(1 L) and extracted with EtOAc (3×150 mL). The EtOAc extracts were dried(MgSO₄), filtered and concentrated to obtain about 8 g of crude residuewhich was crystallized by dissolving in hot CH₃CN (50 mL), followed bydiluting with water (100 mL) and cooling at 0° C. to collect 1.45 g(12.7%) of the title compound as white solid. The filtrate was purifiedby C-18 reverse phase silica gel (YMC ODS-A 75 μm) eluted with 15-30%CH₃CN/H₂O. Appropriate fractions were combined and the aqueous solutionafter removing CH₃CN by rotary evaporator was lyophilized to giveadditional 1.15 g of the title compound 3-81. The crude aqueous layerwas further extracted with EtOAc several times. The ethyl acetateextracts were dried (MgSO4), filtered, concentrated, and crystallizedfrom MeOH to give additional 200 mg of the title compound 3-81. Thetotal yield: 2.8 g (12.2 mmol, Y. 24.5%); MS m/z 230 (MH), HRMS (ESI)m/z calcd for C₁₁H₁₂N₅O (M+H), 230.1042, found 230.1038 (Δ−1.7 ppm); ¹HNMR (CDCl₃) δ ppm 2.54 (3H, s, CH₃), 4.05 (3H, s, OCH₃), 6.73 (1H, s,H-3), 7.40 (1H, s, H-2), 7.56 (1H, s, H-5), 9.15 (1H, s, triazole-H-5);¹³C NMR (CDCl₃, 125.7 MHz) δ ppm 14.2 (triazole-Me), 56.3 (OMe), 100.5(C-3), 116.9 (C-5), 123.5, 127.2, 127.5 (C-2), 129.5 (C-7), 141.2(C-5′), 149.5 (C-4), 161.8 (C-3′); Anal. Calcd for C₁₁H₁₁N₅O: C, 57.63;H, 4.83; N, 30.55; found C, 57.37; H, 4.64; N, 30.68.

The structure was confirmed by a single X-ray crystallographic analysisusing crystals obtained from C-18 column fractions. A portion of C-18column fractions containing a mixture of the desired3-methyl-1,2,4-triazolyl analog 3-81 and isomeric5-methyl-1,2,4-triazolyl analog 4-81 was further purified by C-18reverse phase column eluting with 8-10% CH₃CN/H₂O. Appropriate fractionswere extracted with CH₂Cl₂, and slow evaporation of the solvent gavecrystalline material of the isomeric7-(5-methyl-1,2,4-triazolyl)-4-methoxy-6-azaindole (4-81): MS m/z 230(MH), ¹H NMR (CDCl₃) δ ppm 3.05 (3H, s, CH₃), 4.07 (3H, s, OCH₃), 6.74(1H, q, J=2.4, H-2), 7.37 (1H, t, J=2.4, H-3), 7.65 (1H, s, H-5), 8.07(1H, s, triazole-H-3). The structure was confirmed by a single X-raycrystallographic analysis.

Preparation of 5-81

Procedure:

AlCl₃ (40 g, 0.3 mol, 15 eq.) was dissolved in a solution of CH₂Cl₂ (100mL) and nitromethane (20 mL) under dry nitrogen. To this solution wasadded compound 3-81 (4.58 g, 0.02 mol) under stirring and under N₂,followed by methyl chlorooxoacetate (9.8 g, 0.08 mol, 4 eq.). Themixture was stirred under N₂ at room temperature for 1.5 h. The mixturewas added drop-wise to a cold and stirred solution of 20% aqueousammonium acetate solution (750 mL). The mixture was stirred for 20 minand the resultant precipitate was filtered, washed thoroughly with waterand dried in vacuo to obtain 4.7 g (0.015 mol, Y. 75%) of the titlecompound 5-81 as white solid: MS m/z 316 (MH); HRMS (ESI) m/z calcd forC₁₄H₁₄N₅O₄ (M+H), 316.1046; found 316.1041 (Δ−1.6 ppm); ¹H NMR (CDCl₃,500 MHz) δ ppm 2.58 (3H, s, CH₃), 3.96 (3H, s, OCH₃), 4.05 (3H, s,OCH₃), 7.76 (1H, s, H-5), 8.34 (1H, d, J=3 Hz, H-2), 9.15 (1H, s,triazole-H-5), 11.0 (1H, brs, NH). More title compound 5-81 andhydrolyzed acid 6-81 can be obtained from the filtrate by acid-baseextraction with EtOAc.

Preparation of 6-81

Procedure:

To a suspension of the methyl ester 5-81 (2.2 g, 7.0 mmol) in MeOH (50mL) was added 0.25M NaOH solution in water (56 mL, 14 mmol, 2 eq.) atroom temperature and the mixture stirred for 15 min by which time HPLCindicated the hydrolysis was complete. The mixture was concentrated invacuo quickly to remove MeOH, and to the residual solution was addedwater (100 mL) and 1N HCl (14 mL) with stirring to neutralize themixture. The resultant fine precipitate was filtered, washed with waterand dried in vacuo to obtain 1.98 g (6.58 mmol, Y. 94%) of the titlecompound 6-81 as off-white solid: MS m/z 302 (MH); ¹H NMR (DMSO-d₆, 500MHz) δ ppm 2.50 (3H, s, overlapped with DMSO peaks), 3.98 (3H, s, CH₃O),7.87 (1H, s, H-5), 8.29 (1H, d, J=3.5 Hz, H-2), 9.25 (1H, s,triazole-H-5), 12.37 (1H, s, NH).

Alternative procedure: To a suspension of the methyl ester 5-81 (10.7 g,34 mmol) in MeOH (150 mL) was added 0.25M NaOH solution in water (272mL, 68 mmol, 2 eq.) at room temperature and the mixture stirred for 20min by which time HPLC indicated the hydrolysis was complete. Themixture was concentrated in vacuo quickly to remove MeOH, and theresidual solution was extracted with EtOAc to remove any neutralimpurities. To the aqueous phase was added 1N HCl (68 mL, 68 mmol) toneutralize the product. The resultant mixture was frozen and lyophilizedto obtain 14.1 g (33.7 mmol, Y. 99.2%) of the title compound 6-81,containing 2 mole equivalents of NaCl as off-white solid. This materialwas used in the subsequent reaction without further purification. Thedisodium salt of the title compound 6-81 was obtained by C-18 reversephase column chromatography after sodium bicarbonate treatment: HPLC>97%(AP, uv at 254 nm); HRMS (Na salt, ESI⁻) m/z calcd for C₁₃H₁₀N₅O₄ (M−H),300.0733; found 300.0724 (Δ−3 ppm); ¹H NMR (Na salt, DMSO-d₆, 500 MHz) δppm 2.37 (3H, s, Me), 3.83 (3H, s, CH₃O), 7.56 (1H, s, H-5), 8.03 (1H,s, H-2), 9.32 (1H, s, triazole-H-5); ¹³C NMR (Na salt, DMSO-d₆, 125.7MHz) δ ppm 13.8 (triazole-Me), 57.2 (OMe), 114.8 (C-3), 120.0 (C-5),125.1, 143.5 (C-5′), 149.8 (C-4), 160.0 (C-3′), 171.7, 191.3.

Preparation of Compound IVc

Procedure:

To a solution of the acid 6-81 (3.01 g, 10 mmol) and benzoylpiperazinehydrochloride (3.39 g, 15 mmol) in DMF (50 mL) was added triethylamine(10.1 g, 100 mmol, 10 eq.), followed by1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC; 5.75g, 30 mmol) under N₂ and the mixture stirred at room temperature for 22h after sonication and at 40° C. for 2 h. The mixture was concentratedin vacuo to remove DMF and TEA, and to the residual solution was addedwater (200 mL) under stirring and sonication. The precipitates formedwere collected, washed with water and dried in vacuo to obtain 2.8 g(5.9 mmol, Y. 59%) of the title compound IVc as off-white solid. Thefiltrate was extracted with CH₂Cl₂ (x2). The CH₂Cl₂ extracts were dried(Na₂SO₄), filtered and concentrated to gum which was triturated withEt₂O to obtain a solid. This solid was suspended and triturated withMeOH to obtain 400 mg of the title compound IVc as off-white solid.Total yield: 3.2 g (6.8 mmol, Y. 68%): MS m/z 474 (MH); HRMS (ESI) m/zcalcd for C₂₄H₂₄N₂O₄ (M+H) 474.1890, found 474.1884 (Δ−1.2 ppm); ¹H NMR(DMSO-d6) δ ppm 2.50 (3H, s, overlapped with DMSO peaks), 3.43 (4H, br,CH₂N), 3.68 (4H, br, CH₂N), 3.99 (3H, s, CH₃O), 7.46 (5H, br. s, Ar—Hs),7.88 (1H, s, indole-H-5), 8.25 (1H, s, indole-H-2), 9.25 (1H, s,triazole-H-5), 12.40 (1H, s, NH); ¹³C-NMR (DM50-d6) δ ppm 13.78, 40.58,45.11, 56.78, 114.11, 120.95, 122.71, 123.60, 126.98, 128.34, 129.6,135.43, 138.52, 142.10, 149.15, 161.29, 166.17, 169.22, 185.42; UV(MeOH) λmax 233.6 nm (ε3.43×10⁴), 314.9 nm (ε1.73×10⁴); Anal: Calc forC₂₄H₂₄N₂O₄.⅕H₂O; C, 60.42; H, 4.94; N, 20.55. Found; C, 60.42; H, 5.03;N, 20.65. KF (H₂O) 0.75%.

This reaction can also be performed by use of HATU and DMAP to providemore consistent yield of the title compound: To a suspension of the acid6-81 (15.6 mmol) and HATU[O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphonate] (8.90 g, 23.4 mmol; 1.5 eq.) in DMF (60 mL) and CH₂Cl₂ (60 mL)was added a mixture of DMAP (5.72 g, 46.8 mmol, 3 eq.) andbenzoylpiperazine hydrochloride (5.30 g, 23.4 mmol; 1.5 eq.) in DMF (60mL) at room temperature and the mixture was stirred under nitrogenatmosphere for 4 hrs. The mixture was concentrated in vacuo to removeCH₂Cl₂ and most of DMF, and to the residual solution was added waterunder stirring and sonication. The precipitates formed were collected,washed with water and dried in vacuo to obtain 5.38 g (11.4 mmol, Y.72.8%) of the title compound IVc as off-white solid: HPLC>95% (AP, uv at254 nm).

Synthetic Experimental Procedures for Best Preparation of Compound IVb

5-Amino 2 methoxypyridine (50 g, 0.4 mol) was added to a stirringmixture of absolute ethanol (280 ml) and HBF₄ (48% in water, 172 ml) andcooled to 0° C. Sodium nitrite (129 g) was dissolved in water (52 ml)and added portion-wise over 1 h). The stirring was continued at 0° C.for 2 hr. The reaction mixture was diluted with ether (1 L). The solidproduct was collected by filtration and washed with 500 ml of 50:50EtOH/ether and subsequently several times with ether until the productwas slightly pinkish in color. The pale pink solid 90 g (˜100% yield)was kept in a dessicator over P₂O₅.

The same procedure was followed to perform the reaction on larger scale:

(1) (200 g, 1.6 mol); HBF₄ (688 ml); NaNO₂ (116 g); EtOH (1.12 L); H₂O(208 ml)

The reaction was run 4 times (total 800 grams (1-80)). The product wasdried over P₂O₅ for 48 hr. (only 24 hr for first batch).

A total of 1,293 g of (2-80) was obtained, (91% yield).

-   Ref: J. Heterocyclic Chem., 10, 779, 1973 (for above reactions,    including analytical data)

The decomposition of the diazonium salt was run in 3 batches of:

206 g, 219 g and 231 g using 1.3 L, 1.4 L and 1.6 L of anhydrous toluenerespectively.

The toluene was preheated under nitrogen to 100° C. (internaltemperature) in a 2 L 3-neck round bottom flask provided with amechanical stirrer. The solid was added solid portion-wise via a scoopthrough a powder funnel which was attached to an adapter with slightoutward positive nitrogen flow. During addition, the temperature wasmaintained between 99-102° C. (set at 100° C.) and stirred vigorously.Total addition time was 60 min. for the smaller two batches and 70 min.for the last one. After the addition was finished, each stirringreaction was heated at 110° C. for 1 hr. The heating mantle was removedand stirring was stopped. The reactions were allowed to stand for 2 hr(ambient temp achieved). Safety Note: The reaction contains BF3 soworking with the reaction hot exposes vapors which caused skinirritation with some people. No incidents were noted at ambienttemperature (6 different people). The hot toluene from the reaction waspoured into a 4 L Erlenmeyer (a dark brown oil and residue remained inthe flask). The residue was washed with 50 ml of toluene and poured intothe original toluene extracts.

Add 1.5 L of 1N NaOH to toluene layer, extract and wash with ˜100 ml ofsat aq. NaCl.

Combine NaCl with NaOH layer, re-extract with 150 ml of toluene, washwith 50 ml of sat NaCl.

Combine toluene layers.

Add 1 L of 1N NaOH to residue in reaction flask and swirl to dissolve asmuch residue as possible then add 500 ml Et2O and pour into Erlenmeyer.

Add ˜500 ml more of 1 N NaOH to reaction flask and swirl ˜500 ml ofEt2O.

Combine dark Et2O and NaOH washings in erlenmyer flask.

Et2O/NaOH mixture was poured through powder funnel containing plug ofglass wool to collect dark viscous solid. (Add ˜500 ml more ether towash) into 6 L sep funnel.

Extract. Wash ether layer with ˜200 ml of H₂O and then 100 ml of satNaCl.

Combine all washings with original NaOH aq. Layer and re-extract with500 ml of ether. Wash with 100 ml H₂O and 100 ml of NaCl.

Combine ether extracts. Toluene and ether extracts were checked by LC/MSclean product.

The ether was concentrated on a rotovap and the residue was combinedwith the toluene extracts to make a homogeneous solution which is takento next step as is.

The other two runs were combined and worked up in the same way.

All aqueous layers were checked by LC/MS=no product.

-   Ref: J. Heterocyclic Chem., 10, 779, 1973 (for above reactions,    including analytical data)

A total of 4.6 L of toluene solution containing 3-80 was placed inseveral sealed tubes and treated with 900 ml of 35% HCl at 145° C. for 2hr. LC/MS showed no starting material, only 4. The toluene solution wasdecanted and discarded. The aqueous phase was washed with EtOAc andconcentrated down to remove volatiles to afford a brown solid containingthe desired fluoro-hydroxypyridine 4-80.

A total of 244 g of this solid was collected and taken to next step asis (it was not completely dry).

Note:

We have subsequently run this by decanting the toluene layer first priorto heating to reduce volumes. Same reaction was carried out using HBr(48% in H2O) at 100° C. for 6 h with similar result to the literatureprocedure 49% yield.

-   Ref: J. Heterocyclic Chem., 10, 779, 1973 (for above reactions,    including analytical data)

The solid from above containing (4-80) was divided in 4 batches andtreated with H₂SO₄ and fuming HNO₃ as shown below. The amounts usedwere:

batch 1 batch 2 batch 3 batch 4 (1) 25 g 54 g 75 g 90 g fuming HNO₃ 20.8ml 45 ml 62.4 ml 75 ml H₂SO_(4 .(for addition)) 5.6 ml+ 12 ml+ 16.8 ml+20 ml+ (for soln) 56 ml 120 ml 168 ml 200 ml

Compound 4-80 was dissolved in sulfuric acid (the larger amountsindicated above) at rt and then heated to 65° C. A preformed solution offuming nitric acid and sulfuric acid (the smaller amount indicatedabove) was added dropwise. The temperature was kept between 65° C. and80° C. (r×n is exothermic and although the bath is at 65° C.,temperature goes higher, usually 75, sometimes 80° C.). After theaddition was complete, the reaction mixture was heated at 65° C. for anadditional hr. The reaction mixture was then cooled to rt and poured ina flask containing ice) (20 g of ice/gr compound, evolution of gasoccurred). A solid precipitated out and it was collected by filtration(¹HNM” showed 4-80 and something else (discarded)).

The aqueous layer was extracted with AcOEt several times (3-5) andconcentrated on a rotary evaporator under vacuum to afford a solid thatwas triturated with ether to afford 5-80 as a bright yellow solid. Atotal of 117 g of desired product was collected in the first crop (27%yield from diazonium salt). A portion did not crystallize: this oil wastriturated with MeOH and Et₂O to afford 3.6 g of 5-80; anotherprecipitation from the mother liquid afforded an additional 6.23 g ofthe desired product 5-80.

Total: 117.0+3.6+6.23=126.83. 30.4%). Yield for 3 steps (decompositionof diazonium salt; deprotection and nitration).

Analytical data from Notebook: 53877-115: ¹HNMR (δ, MeOD): 8.56-8.27(dd, J=7.5, 3.3 Hz, 1H), 8.01 (d, J=3.3 Hz, 1H); LC/MS (M+1)⁺=158.9;rt=0.15 min.

Note:

A portion of the aqueous acidic solution was taken and neutralized withNa₂CO₃ until effervescence stopped and then it was extracted with AcOEt

A different product was obtained. No desired product in these extracts.

A total of 117 g of 5-80 was divided in 4 batches of 30 g×3 and 27 g×1and treated with POBr₃ (3 equiv.; 163 g×3 and 155 g×1) and a catalyticamount of DMF (15 ml) at rt (DMF was added carefully

gas evolution). After 5 min. at room temperature, the solutions wereheated at 110° C. for 3 hr. LC/MS showed starting material had beenconsumed. The reaction mixtures were allowed to cool to rt. The reactionflasks were placed in an ice bath; and then ice was added very slowlyand carefully portionwise into the flask, gas evolution was due to HBrformation; the liquid and black solid that formed was poured into abeaker with ice. EtOAc was added and the mixture was then extractedseveral times with EtOAc. The organic layer was washed with saturatedaq. NaHCO₃; H₂O and brine; dried over Na₂SO₄ and filtered. The productwas dried in the pump overnight to provide 123 g of 6-80 as a brownsolid (77% yield).

Note:

Reaction is completed within 1 h.

¹HNMR (δ, CDCl₃): 8.52 (m, 1H), 7.93 (m, 1H).

800 ml of vinyl magnesium bromide (1M in THF, Aldrich) was cooled below−60° C. with vigorous stirring under N₂. 2-bromo-5-fluoro-3-nitropyridine (43.3 g, 0.196 mol) in 200 ml THF was added dropwise viaaddition funnel at such a rate that the temp was kept below −60° C. Thistook ˜1.25 hr. The reaction mixture was warmed to −40 to −50° C. andstirred for 1 hr more. Then 1 L of saturated aqueous NH₄Cl was addedslowly and cautiously. At first, foaming occurred and considerable solidwas present, but this essentially dissolved as the addition wascompleted and the material warmed to rt. The layers were separated andthe aqueous layer extracted 3 times with ethyl acetate. The organicextracts were washed with brine, dried over Na₂SO₄, filtered andconcentrated to afford ˜50 g of a black gummy solid. HPLC indicated57-58% product. To this was added CH₂Cl₂ and the solid was collected byfiltration and washed with CH₂Cl₂ to afford 12.5 g of product as a brownsolid. The reaction was repeated on exactly the same scale and worked upin the same manner. From CH₂Cl₂ trituration there was obtained 12.4 g ofPrecursor 2i (HPLC ˜97% pure). The crude was recovered and allowed tostand in dichloromethane. Upon standing 3.6 g of additional productseparated and was recovered by filtration.

Total yield=29.5 g (35%).

¹HNMR (δ, CDCl₃): 8.69 (bs, 1H), 7.92 (d, J=1.8 Hz, 1H), 7.41 (m, 1H),6.77 (m, 1H); LC/MS (M+1)⁺=216.−217.9; rt=1.43 min.

Reaction was carried in a 250 ml flask (foaming occurred upon heatingand the big size flask is more convenient). A mixture of precursor 2i (3g, 13.95 mmol), 1,2,3-triazole (15 g, 217.6 mmol, 15 eq), K₂CO₃ (1.9 g,13.95 mmol, 1 eq) and Cu(0) (0.9 g, 13.9 mmol, 1 eq) was heated at 160°C. for 7 hr (from rt to 160° C. total 7 hr) under N₂ (depending on theCu(0) lot, reaction time may vary from 2 hr to 7 hr). The resultingmixture was diluted with MeOH, filtered through filter paper (to removethe copper). Washed with MeOH (20 ml) and water (30 ml).

The filtrate was concentrated (remove solvent in rotovap) and dilutedwith ethylacetate. The aqueous layer was extracted with ethylacetate.The combined organic layer was dried over sodium sulfate, filtered andconcentrated. The residue was dissolved in MeOH (20 ml), 7-80 (750 mg)crystallized from the methanol as a white solid and was collected byfiltration. (Slow gradient volume, silica gel hex/AcOEt (0→18%) of themother liquids usually affords 5-10% more of 7-80.

¹HNMR (δ, CDCl₃): 10.47 (bs, 1H), 8.76 (s, 1H), 7.94 (s, 1H), 7.89 (s,1H), 7.53 (m, 1H), 6.78 (m, 1H); LCMS (M+1)⁺=204; rt=1.29 min.

Ethyl methylimidazolium chloride (4.3 g, 29.6 mmol, 3 eq) was placed ina 250 ml flask. AlCl₃ (11.8 g, 88.6 mmol, 9 eq) was added into the flaskin one portion. A liquid suspension was formed (some of AlCl₃ remainedas solid). After stirring for 5-10 min. compound (1) (2.0 g, 9.85 mmol)was added in one portion followed by slow addition (via a syringe) ofethyl chlorooxalacetate (3.3 ml, 29.6 mmol, 3 eq). The reaction wasstirred at room temperature for 20 hr. LCMS indicated compound8-80:compound 7-80=6:2. (Compound I has strong UV absorption). Thereaction was quenched by carefully adding ice water (˜75 ml) at 0° C. Ayellow solid precipitated at this point. The resulting suspension wasfiltered and the solid was washed with water. MeOH and ethyl acetate (toremove unreacted SM) and the solid was dried in air. (LCMS purity70%˜80%) 2 g of solid containing 8-80 was obtained and taken to the nextstep without further purification. LCMS (M+1)⁺=276; rt=0.97 min.

A mixture of compound 8-80 (4.9 g, 17.8 mmol) & N-benzoylpiperazinehydrochloride 8a-80 (HCl salt; 6.0 g, 26.7 mmol, 1.5 eq) in DMF (30 ml)was stirred at RT overnight (16 hr). A slurry was formed. An additional20 ml of DMF was added into the slurry. Then HATU (12.2 g, 26.7 mmol,1.5 eq) was added followed by DMAP (4.3 g, 35.6 mmol, 2 eq). Thereaction mixture was stirred for 30 min. LCMS indicated the startingmaterial 8-80 was completely converted to product (EXAMPLE 216). Theresulting mixture was filtered and the solid washed with water. Thefiltrate was concentrated in vacuo. Water was added to the residue andthe solid was collected by filtration. The solids were combined andwashed with water, MeOH and EtOAc. Then the solid was dried in air. LCMS& HPLC showed IVb, >99% pure. The solid product was further purified byprecipitation and crystallization in 5˜10% CH₃OH/CHCl₃.

Purification of IVb

Crude compound IVb obtained as above (15.3 g) was dissolved in 10%MeOH/CHCl₃ (600 ml). A light brown suspension was formed, filteredthrough filter paper and washed with MeOH a twice. The brownish solidwas discarded (˜1.2 g). Compound IVb was crystallized in the filtrate,the solid was collected by filtration and the white solid was dried inair. The filtrate was used to repeat the crystallization several times.The solid obtained from each filtration was analyzed by HPLC. All thepure fractions were combined. The not so pure fractions were resubjectedto crystallization with MeOH & CHCl₃. A total of 12.7 g of Compound IVbwas obtained from recrystallization and precipitation. The mother liquidwas concentrated and purified on silica gel column (EtOAc, thenCHCl₃/MeOH (0-2%)) to provide 506 mg of product) as a white solid.

¹HNMR (d, DMSO) 13.1 (bs, 1H), 9.0 (s, 1H), 8.4 (s, 1H), 8.3 (s, 1H),8.2 (s, 1H), 7.4 (bs, 5H), 3.7 (bs, 4H), 3.5 (bs, 4H); MS m/z 448 (MH).Anal: Calc for C₂₂H₁₈FN₇O₃; C, 59.05; H, 4.05; N, 21.91; F, 4.24. Found;C, 57.28; H, 4.14; N, 21.22; F, 4.07%.

Examples 1-4 Preparation of Prodrugs I from Parent Compounds IVSynthetic Scheme for Examples 1-4

Example 1 Preparation of Ia

Procedure:

A suspension of IVa (211 mg, 0.5 mmol) in THF (2 mL; Sure Seal) underanhydrous N₂ atmosphere was treated with NaH (86 mg, 2.2 mmol; 4.4 eq.;60% oil dispersion). After a few minutes stirring at room temperature,di-tert-butyl chloromethyl phosphate (782 mg, 3.0 mmol; preparation, seeU.S. Pat. No. 6,362,172) was added and the mixture stirred for 1-5 days,monitoring the completion of the reaction by HPLC (additional 1-2 eq. ofeach NaH and the phosphate may be required to bring the reaction to nearcompletion). After the staring material was consumed, the mixture wasconcentrated in vacuo to dryness and the residue, which appeared to be amixture of N-alkylated indole mono- and bis-t-butyl phosphate, wasdissolved in CH₂Cl₂ (5 mL) was treated with TFA (5 mL) at roomtemperature for 2 h. The mixture was concentrated in vacuo and theresidue was purified by C-18 reverse phase silica gel, eluting with5-10% CH₃CN in water containing NaHCO₃, to obtain 75 mg (0.13 mmol; Y.26%) of the title compound Ia as an off-white powder (disodium salt):HPLC>99% (AP at 254 nm); LC/MS (ESI+) m/z 533 (M+H minus 2Na)⁺; HRMS(ESI) m/z calcd for C₂₃H₂₆N₄O₉P (M+H minus 2Na)⁺ 533.1437, found533.1426 (Δ−2.1 ppm); ¹HNMR (D₂O, 500 MHz) δ ppm 3.51 (2H, m), 3.67 (2H,m), 3.73-3.79 (2H, m), 3.89-3.95 (2H, m), 3.94, 3.95 (3H, 2s), 4.05,4.07 (3H, 2s), 6.02-6.04-6.05-6.07 (2H, ABq.), 7.43, 7.44 (1H, 2s),7.45-7.56 (5H, m), 8.49, 8.52 (1H, 2s).

By using a similar procedure and conditions, Ib was prepared from IVb.Ic and Id were prepared from IVc, and IVd, respectively, but waterrather than sodium bicarbonate solution was utilized in thepurification.

Example 2

Ib: Yield 13% (disodium salt); HPLC>96% (AP at 254 nm); LC/MS (ESI+) m/z558 (M+H minus 2Na)⁺; ¹H NMR (D₂O, 500 MHz) δ ppm 3.59 (2H, m),3.70-3.84 (4H, m), 3.93-3.95 (2H, m), 5.28-5.29-5.30-5.32 (2H, ABq.),7.4-7.6 (5H, m), 8.09, 8.10 (1H, 2s), 8.34, 8.36 (1H, 2s), 8.59, 8.61(1H, 2s), 8.72, 8.75 (1H, 2s).

Example 3

Ic: Yield 37% (acid form. Water was used in place of aqueous sodiumbicarbonate during the purification; HPLC>98% (AP at 254 nm); LC/MS(ESI+) m/z 584 (M+H); ¹H NMR (DMSO-d6, 500 MHz) δ ppm 2.40 (3H, s), 3.44(4H, br.s), 3.66 (4H, brs), 4.04 (3H, s), 5.79 (1H, s), 5.82 (1H, s),7.46 (5H, brs), 8.07 (1H, s), 8.41 (1H, s), 8.88 (1H, s).

Example 4

Id: Yield 7% (acid form). Water was used in place of aqueous sodiumbicarbonate during the purification; LC/MS (ESI+) m/z 597 (M+H); ¹H NMR(DMSO-d6, 500 MHz) δ ppm 1.16, 1.21 (3H, 2d, J=6.5 Hz), 2.29 (3H, s),2.3-4.5 (7H, m), 4.00, 4.01 (3H, 2s), 5.79-5.85 (2H, m), 6.36 (1H, t,J=2 Hz), 7.42-7.47 (5H, m), 7.99 (1H, s), 8.08, 8.09 (1H, 2s), 8.34,8.44 (1H, 2s).

Example 5 Preparation of Ica, (Disodium Salt)

General Procedure:

A suspension of IVc (0.24 g, 0.5 mmol) in anhydrous THF (4 mL) undernitrogen atmosphere was treated with sodium hydride (60% oil dispersion,0.08 g, 2.0 mmol), and stirred until gas evolution ceased (approximately5 minutes). The reaction mixture was treated with iodine (0.13 g, 0.5mmol) and stirred for 2-3 minutes followed by addition of di-tert-butylchloromethyl phosphate (1.6 g, 6.0 mmol, crude). A stream of nitrogenwas allowed to pass over the reaction to facilitate the removal of muchor all of the THF. The reaction mixture was stirred overnight. HPLCanalysis of crude indicated starting IVc (ca. 56%) and desired adduct(ca. 32%).

Several crude reaction mixtures (a total of 6.7 mmol based on startingmaterial IVc) were re-dissolved in dichloromethane, combined,concentrated in vacuo to remove any remaining THF. The residue wassuspended in dichloromethane and TFA (1:1, approximately 40 mL totalvolume). The mixture was stirred for 1.5-2 hours and then solvent wasremoved in vacuo. The residue was suspended in dichloromethane andextracted into water (approximately 60 mL) made weakly basic with solidor aqueous sodium bicarbonate. The aqueous layer was reduced in volumeby rotary evaporator if required and the solution was loaded onto a C-18reverse phase column (approximately 80 g of C-18, YMC ODS-Aq, 50 micron)and eluted with water, followed by water containing 2.5% acetonitrile.Fractions containing pure product were pooled and organic solvent wasremoved by rotary evaporator. Purified product was recovered afterlyophilization to give 1.00 g (1.30 mmol, 19% over 2 steps) of the titlecompound Ica (disodium salt) as an off-white powder: HPLC purity>99% APat 254 nm (gradient 0-100% B/A; A 10% CH₃CN—90% H₂O—0.1% TFA, B 90%CH₃CN—10% H₂O—0.1% TFA, gradient time 4 min, column YMC ODS-Aq 4.6×50 mm3 micron); MS-ESI− m/z 482 (M−H minus 2Na)⁻; HRMS (ESI) m/z calcd forC₂₅H₂₇N₇O₈P (M+H minus 2Na)⁺ 584.1659, found 584.1651 (Δ−1.3 ppm); ¹HNMR (D₂O, 500 MHz) δ ppm 2.53, 2.54 (3H, 2s), 3.56 (2H, s, CH₂N), 3.72(2H, br.s, CH₂N), 3.78, 3.83 (2H, 2br.s, CH₂N), 3.94, 3.96 (2H, 2 br.s,CH₂N), 4.14 (3H, s, CH₃O), 5.38, 5.40 (2H, 2d, J=11 Hz), 7.45-7.59 (5H,m, Ar—Hs), 8.07, 8.09 (1H, 2s, indole-H-5), 8.64, 8.67 (1H, 2s,indole-H-2), 8.87, 8.89 (1H, 2s, triazole-H-5); ¹³C-NMR (125.7 MHz, D₂O)δ ppm 15.43 (N-Me), 44.03, 44.47, 44.66, 45.05, 48.20, 48.82, 49.60,50.23, 59.78 (OMe), 75.81 (NCH₂O), 115.6, 126.0, 127.2, 129.6, 131.0,131.7, 132.1, 133.5, 136.8, 147.6, 150.1, 154.2, 164.8, 170.4, 175.8,189.2; UV (H2O) λmax 220 nm (ε3.91×10⁴), 249 nm (ε2.00×10⁴), 303 nm(ε1.60×10⁴); Anal: Calc for C₂₅H₂₄N₇O₈PNa₂. 8H₂O. 0.2NaHCO₃; C, 38.39;H, 5.14; N, 12.44; P, 3.93; Na, 6.42. Found; C, 38.16; H, 4.81; N,12.43; P, 3.72; Na, 6.05. KF (H₂O) 17.3%.

A less pure fractions were collected to obtain 0.22 g (0.29 mmol, Y. 4%)of the title compound Ica (disodium salt): HPLC purity>95% (AP at 254nm).

Example 6 Preparation of Iab (Hydrated Lysine Salt) Step One

Phosphate ester A (45.1 g, 0.1 mol) and chloroiodomethane B (200 g, 1.14mol) were combined in 100 ml of benzene and the mixture was stirred atroom temperature for four hours before benzene was removed under vacuum.Then, 500 ml of ethyl ether was added to the residue and insoluble solidwas filtered away. Concentration of the filtrate provided di-tert-butylchloromethyl phosphate, which was utilized in the next step without anypurification.

Step Two

NaH (2.4 g, 60% in oil) was added slowly into a suspension of IVa in dryTHF (120 ml) and the mixture was allowed to stir for an hour at roomtemperature. Iodine (5 g) dissolved in dry THF (10 ml) was added slowlyinto the stirring solution. Following completion of addition theresultant mixture was stirred at ambient temperature for an additional15 minutes and then compound di-tert-butyl chloromethyl phosphate,obtained from step one, was added. After stirring for 16 hours, thereaction mixture was poured into ice water (120 ml), followed byextraction with EtOAc (3×300 ml). The combined organic extracts werewashed with water (100 ml) and then brine (100 ml), dried over Na₂SO₄,and concentrated under vacuum to afford a residue, which was purified bysilica gel chromatography (elution with EtOAc/Et₃N (100/1) and thenEtOAc/MeOH (100/1)) to give diester IIa in yields of 70-80%.

Step Three

A mixed solution of TFA (50 ml) and dichloromethane (450 ml) was addedinto a round bottom flask containing 43.3 g of diester Ha. Afterstirring at room temperature for 16 hours, the reaction mixture wasconcentrated under vacuum to offer a residue of Iac which was used infurther steps without any purification.

Step Four

The above 55 g crude product Iac was added to an aqueous solution ofL-lysine (1.36M, 70 mL) at room temperature. The resulting suspension(pH=1.83) was added to a lysine solution (1.36 M, ˜40 mL) to pH 4.88.The resulting suspension was filtered through a pad of Celite. The clearlight yellow filtrate (˜200 mL) was mixed with acetone (200 mL) andheated to 45° C. Acetone (1400 mL) was added over 2 h at 45° C. Theclear solution was seeded and stirred at 45° C. for 2 h, and slowlycooled to room temperature (5 h) and the suspension stirred overnight.The white solid was collected by filtration and dried under house vac.at 50° C. over 24 h to afford 41.2 g of Iab as an off-white solid.

The above solid was dissolved in 1:1 water-acetone (560 mL) at 45° C.Acetone (700 mL) was added over a period of 1 h at 45° C. The clearsolution was seeded and stirred at 45° C. for 2 h. Slowly cooled to roomtemperature (5 h) and the suspension stirred at room temperatureovernight. The white solid was collected by filtration and dried underhouse vac. at 50° C. over 36 h to afford 33 g of Iab as an off-whitesolid. The AP was >99% by HPLC.

¹H NMR (500 MHz, D₂O) δ8.42 (s, ½H), 8.39 (s, ½H), 7.52 (m, 6H), 6.12(m, 2H), 4.07 (s, 3H), 3.93 (m, 5H), 3.72 (m, 3H), 3.67 (m, 2H), 3.52(m, 2H), 3.05 (m, 2H), 1.93 (m, 2H), 1.74 (m, 2H), 1.50 (m, 2H); MS m/z:(M+H-lysine)⁺ calcd for C₂₃H₂₆N₄O₉P 533.14, found 533.03. M.P. 166.7 to172.2 degrees. Using comparative ¹H NMR integration of several differentpeaks, the ratio of lysine to IVa is calculated to range from 1.05:1 to1.2:1 equivalents of lysine to parent prodrug. The salt form wasdetermined to be a hydrate. Based on DSC (diffraction scanningcalorimetry) and TGA (thermal gravity analysis), the observed watercontent is 2.80%. Theoretical calculation for a monohydrate is 2.58%.Thus, the ratio of water to parent molecule in the hydrate could be inthe range 1:1 to ˜1.5:1.

Example 7 Preparation of Crystalline Ic (Free Acid Mono-Hydrate)

To a mixture of IVc (600 mg, 1.27 mmol) in anhydrous THF (10 ml) in anoven-dried round bottle flask under nitrogen at r.t. was added NaH (153mg, 6.38 mmol, dry powder, 95%), and the white suspension stirred untilno gas evolution was observed. The mixture was then added I₂ (375 mg,1.48 mmol), and stirred at r.t. for 3 h. To the reaction mixture wasadded NaH (153 mg, 6.38 mmol, dry powder, 95%), and the mixture stirredfor about 5 to 10 min. The crude chloromethyl di-tert-butylphosphate(2.0 g, about 1.6 ml, 7.79 mmol) was added to the mixture, which wasthen stirred at r.t. for 15 h. LCMS analysis of the reaction showeda >97% conversion of the starting material. After evaporation of thevolatiles, the residue was added CH₂Cl₂ (10 ml), cooled in an ice-waterbath, slowly added TFA (10 ml) and stirred at r.t. for 3 h. The reactionmixture was then evaporated, and the residue partitioned between CH₂Cl₂(50 ml) and H₂O (50 ml). The CH₂Cl₂ layer was poured into the reactionflask that contained some undissolved brownish solid, and this mixturewas extracted with a dilute aqueous NaHCO₃ solution (50 ml). The aqueousmixture was purified by reverse phase preparative HPLC (solvent A: 10%MeOH—90% H₂O—0.1% TFA; solvent B: 90% MeOH—10% H₂O—0.1% TFA; start %B=0, final % B=100; gradient time=6 min; flow rate=45 ml/min; column:phenomenex-Luna 30×50 mm, S5; fraction collected: 3.65 to 4.05 min) Thefractions collected were evaporated to dryness, and the residue driedunder high vacuum to obtain the acid Ic as a pale yellow solid (356.6mg); ¹H NMR: (500 MHz, CD₃OD) δ 9.05 (s, 1H), 8.46 (s, 1H), 8.04 (s,1H), 7.47 (bs, 5H), 5.93 (d, J=12, 2H), 4.10 (s, 3H), 4.00-3.40 (bs,8H), 2.53 (s, 3H); ¹⁹F NMR analysis showed that the material containedresidual TFA, (the percentage was not quantified); Analytical HPLCmethod: Start % B=0, Final % B=100, Gradient time=2 min, Flow Rate=5mL/min, Column: Xterra MS C18 7u 3.0×50 mm, LC/MS: (ES+) m/z (M+H)⁺=584,HPLC R_(t)=0.983.

172.2 mg of the purified acid Ic was dissolved in 1 ml of H₂O and thenabout 0.3 ml of absolute EtOH (200 proof) was added. The mixture wasleft standing in a refrigerator (temperature about 3° C.) overnight,after which time, crystalline material was observed. The mixture wasthen warmed to ambient temperature, diluted with H₂O to a volume of 3mL, and then 20 mL of MeCN was added slowly. Following the completion ofaddition, the mixture was stirred at r.t. for 2 h and then filtered. Thesolid collected (90 mg) was dried in vacuo, and then under high vacuum.This material was shown by powder x-ray studies to be crystalline;Elemental Analysis calculated for C₂₅H₂₆N₇O₈P.H₂O: C, 49.92; H, 4.69; N,16.30; observed: C, 49.66; H, 4.62; N, 15.99. mp=205° C. (measured bydifferential scanning calorimetry). The ¹H NMR pattern for crystallinematerial was compared with that from the purified acid and both wereconsistent with the structure.

Example 8 Preparation of Iab (Mono L Lysine Salt):{3-[(4-benzoylpiperazin-1-yl)(oxo)acetyl]-4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-1-yl}methylDihydrogen Phosphate, L-lysine Salt (1:1)

The sequence of reactions is described in Scheme for Example 8.

Scheme for Example 8

Preparation of di-tert-butyl Chloromethyl Phosphate

The tetrabutylammonium salt of bis-tert butyl phosphate (45.1 g, 0.1mol) and chloroiodomethane (200 g, 1.14 mol) were combined in 100 ml ofbenzene and the mixture was stirred at room temperature for four hoursand then the benzene was removed under vacuum. A portion of 500 ml ofethyl ether was added to the residue and insoluble solid was filteredaway. Concentration of the filtrate in vacuo and removal of thevolitiles on a vacuum pump provided di-tert-butyl chloromethylphosphate, as a light yellow or light brown oil which was utilized inthe next step without further purification.

Preparation of IIa:(3-(2-(4-benzoylpiperazin-1-yl)-2-oxoacetyl)-4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-1-yl)methyldi-tert-butyl Phosphate

NaH (2.4 g, 60 mmol, 60% in oil) was added slowly to a suspension of(8.4 g, 20 mmol) IVa in dry THF (120 ml) and the mixture was allowed tostir for an hour at room temperature. Iodine (5 g, 20 mmol) dissolved indry THF (10 ml) was added slowly and cautiously to the stirring solutionat a rate to keep foaming under control. Following completion ofaddition, the resultant mixture was stirred at ambient temperature foran additional 15 minutes and then the ˜0.1 mol di-tert-butylchloromethyl phosphate, obtained as described in step one, was added.After stirring for 16 hours, the reaction mixture was poured into icedNH₄OAc (30%) (120 ml), followed by extraction with EtOAc (3×300 ml). Thecombined organic extracts were washed with water (100 ml) and then brine(100 ml), dried over Na₂SO₄, and concentrated invacuo to afford aresidue, which was purified by silica gel chromatography (elution withEtOAc/Et₃N (100/1) and then EtOAc/MeOH (100/1) to give diester IIa(9.0-10.3 gs, AP ˜75%) as a light yellow solid in yields of 70-80% overseveral runs.

¹H NMR (500 MHz, CDCl₃) δ8.09 (s, 1H), 7.48 (s, 1H), 7.40 (b, 5H), 6.15(d, 2H, J=11.5 Hz), 4.05 (s, 3H), 3.90 (s, 3H), 3.90-3.30 (b, 8H), 1.39(s, 18H); ¹³C NMR (125 MHz, CDCl₃) δ185.5, 170.7, 166.5, 146.9, 146.2,139.6, 135.3, 130.2, 128.7, 128.4, 127.2, 124.5, 122.0, 120.8, 115.8,83.8, 73.2, 57.3, 53.5, 46.1, 41.7, 29.8; MS m/z: (M+H)⁺ calcd forC₃₁H₄₂N₄O₉P 645.27, found 645.10.

Preparation of Iab:{3-[(4-benzoylpiperazin-1-yl)(oxo)acetyl]-4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-1-yl}methylDihydrogen Phosphate, L-lysine Salt (1:1)

500 mg of diester Ha was dissolved in a mixture of water (3 ml) andacetone (3 ml). The resulting mixture was stirred at 40° C. for 16 hoursto allow the solvolysis to reach completion. To this reaction mixture(˜69 AP) was added 4M aqueous lysine solution to adjust pH to 4.83.Acetone (35 ml) was slowly added into the reaction mixture in 30 min at45-50° C. At 45° C., the clear solution was seeded with crystalline Iaband kept stirred at this temperature for 45 min. After complete additionof acetone, the solution was cooled to room temperature in 4 hours andthe crystallization of Iab completed overnight. The solid was collectedby filtration and suction under nitrogen for 2 hours. The whitecrystalline solid was dried under house vacuum at 50-55° C. for 24 h toafford 343 mg of Iab.

Iab obtained in the above operation: ¹H NMR (500 MHz, CD₃OD) δ8.38 (s,1H), 7.49 (m, 6H), 6.13 (d, 2H, J=10.5 Hz), 4.06 (s, 3H), 3.92 (s, 3H),4.00-3.40 (m, 8H), 3.58 (t, 1H, J=6 Hz), 2.92 (t, 2H, J=7.5 Hz),1.90-1.40 (m, 6H); ¹³C NMR (125 MHz, CD₃OD) δ186.1, 173.2, 171.8, 167.8,147.4, 146.4, 141.0, 135.4, 130.4, 128.8, 127.2, 124.6, 122.3, 120.2,114.6, 73.2, 56.6, 54.7, 53.1, 46.0, 41.6, 39.2, 30.5, 27.0, 22.0. HRMSm/z: (M-lysine+H)⁺ calcd for C₂₃H₂₆N₄O₉P 533.1437, found 533.1437. Anal.Calcd. C, 51.32; H, 5.79; N, 12.38; P, 4.56; found: C, 48.54; H, 5.32;N, 11.76; P, 4.04. Melting Point 170° C.

Obtained via other process (hydrolysis with TFA in methylene chloride),Iab was a 1.70 molar hydrate and 1.14 molar lysine salt. ¹H NMR (500MHz, D₂O, 60° C.) δ8.72 (s, 1H), 7.84 (m, 6H), 6.44 (d, 2H, J=10 Hz),4.41 (s, 3H), 4.27 (s, 3H), 4.3-3.7 (m, 8H), 4.10 (t, 1H, J=5 Hz), 3.39(t, 2H, J=5 Hz), 2.30-1.80 (m, 6H); ¹³C NMR (125 MHz, D₂O, 27° C.)δ186.7, 174.9, 173.2, 167.9, 147.7, 145.7, 142.6, 134.3, 131.1, 129.2,127.1, 124.3, 122.4, 120.1, 113.8, 73.5, 57.1, 54.9, 54.4, 47.7, 47.1,46.3, 45.7, 42.6, 42.1, 42.0, 41.5, 39.5, 30.2, 26.8, 21.8. HRMS m/z:(M−lysine+H)⁺ calcd for C₂₃H₂₆N₄O₉P 533.1437, found 533.1425. Anal.Calcd. C, 49.11; H, 6.13; N, 12.05; found: C, 48.93; H, 6.26; N, 12.07.M.P. 168-172° C.

Example 9 Preparation of Ibb (Mono L Lysine Salt):[3-[(4-benzoylpiperazin-1-yl)(oxo)acetyl]-4-fluoro-7-(1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl]methylDihydrogen Phosphate, L-lysine Salt (1:1)

The sequence of reactions is described in the Scheme for Example 9.

Scheme for Example 9

Preparation of di-tert-butyl Chloromethyl Phosphate

The tetrabutylammonium salt of bis-tert butyl phosphate (57 g, 0.126mol, Digital Specialty Chemicals) and chloroiodomethane (221 g, 1.26mol) were stirred at room temperature for four hours and then thevolatiles were removed in vacuo. 500 ml of ethyl ether was added to theresidue and insoluble solid was filtered away. Concentration of thefiltrate and final removal of volatiles using a vacuum pump provideddi-tert-butyl chloromethyl phosphate (112 g), typically as a lightyellow or brown oil, which was utilized in the next step without anyfurther purification.

Preparation of IIb:(3-(2-(4-benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluoro-7-(1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methyldi-tert-butyl Phosphate

NaH (5.65 g, 95% dispersion in mineral oil, 0.224 mol) was added slowlyinto a suspension of IVb (20 g, 44.7 mmol) in dry THF (400 ml) and themixture was allowed to stir for 0.5 hour at room temperature. A solutionof iodine (11.3 g, 44.5 mmol) dissolved in dry THF (20 ml) was addedslowly into the stirring solution at a rate which kept the reaction frombecoming violent. The resultant mixture was stirred for an additional 3hours before a second portion of 95% NaH (5.65 g, 0.224 mol) wasintroduced. After 15 minutes at ambient temperature, di-tert-butylchloromethyl phosphate, (112 g), obtained from step one, was added inone portion. After stirring for 16 hours at ambient temperature, thereaction mixture was poured into iced NH₄OAc (30%) (200 ml) and thenextracted with EtOAc (3×500 ml). The combined organic extracts werewashed with water (200 ml) and then brine (200 ml), dried over Na₂SO₄,and concentrated under vacuum to afford a residue, which was purified bysilica gel chromatography (elution with EtOAc/MeOH/Et₃N (100/1/1) togive 15.0 gs (43% yield corrected for 85% AP) of diester IIb as a lightyellow solid.

¹H NMR (500 MHz, CDCl₃) δ8.36 (s, 1H), 8.25 (s, 1H), 8.21 (s, 1H), 7.88(s, 1H), 7.41 (b, 5H), 5.90 (d, 2H, J=14.5 Hz), 3.90-3.40 (b, 8H), 1.23(s, 18H); ¹³C NMR (125 MHz, CDCl₃) δ182.9, 170.7, 165.1, 154.6, 152.5,144.1, 135.1, 134.0, 131.9, 130.3, 128.7, 128.3, 127.2, 125.9, 124.3,114.0, 84.1, 74.1, 46.2, 41.9, 29.6; HRMS m/z: (M+H)⁺ calcd forC₃₁H₃₈FN₇O₇P 670.26, found 670.34.

Preparation of Ibb (Mono L Lysine Salt):[3-[(4-benzoylpiperazin-1-yl)(oxo)acetyl]-4-fluoro-7-(1H-1,2,3-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl]methylDihydrogen Phosphate, L-lysine Salt (1:1)

Diester IIb (27 g) was dissolved in a mixture of water (55 ml) andacetone (55 ml). The resulting mixture (pH: not determined) was stirredat 40° C. for 16 hours to complete the solvolysis. To this reactionmixture was added 4M aqueous lysine solution to adjust pH to 3.51. EtOH(500 ml) was added into the solution and the flask wall was coated withsome product after overnight. The clear solution was then transferred toanother flask and EtOH (1500 ml) was slowly added to the reactionmixture in ˜3 h. After complete addition of ethanol, the solution wasstirred at room temperature for 48 hours and the resultant solid (Ibb)was collected by filtration and rinsed with ethanol. The whitecrystalline solid was dried under house vacuum at 55° C. for 24 h toafford 10.92 g of Ibb (98 AP).

This solid was further mixed with 12.5 g of salt obtained from otheroperations in 70 ml of water. EtOH (1000 ml) was then added and theresultant solution was stirred at r.t. for over 20 hours. The solid wascollected by filtration, rinsed with EtOH (2×80 ml) and dried underhouse vacuum at 50° C. under nitrogen atmosphere for 44 hours to afford21.5 g of Ibb in AP of 98.7.

Ibb obtained in the above procedure was ˜1 molar lysine salt with 1.12%of water, 0.8% of TFA and 0.05% of ethanol. ¹H NMR (500 MHz, CD₃OD, 50°C.) δ8.94 (s, 1H), 8.87 (s, 1H), 8.69 (s, 1H), 8.42 (s, 1H), 7.83 (m,5H), 5.81 (d, 2H, J=12.5 Hz), 4.30-3.70 (m, 8H), 4.08 (t, 1H, J=6.5 Hz),3.67 (t, 2H, J=10 Hz), 2.26 (m, 2H), 2.07 (m, 2H), 1.88 (m, 2H); ¹³C NMR(125 MHz, D₂O, 30° C.) δ185.2, 174.9, 173.3, 166.8, 153.1, 146.8, 134.8,134.3, 131.3, 131.1, 130.3, 129.3, 128.9, 128.7, 128.5, 127.2, 124.2,112.6, 74.0, 54.9, 47.9, 47.2, 46.4, 45.9, 42.7, 42.2, 42.0, 41.7, 39.5,30.3, 26.8, 21.8. MS m/z: (M-lysine+H)⁺ calcd for C₂₃H₂₂FN₇O₇P 558.1302,found 558.1293. Anal. Calcd. C, 48.75; H, 5.07; N, 17.63; P, 4.33;found: C, 49.02; H, 4.90; N, 17.90; P, 4.37. M.P. 193° C. pKa(potentiometric) 6.1, 9.1.

Example 10 Preparation of Icb (Mono Tromethamine Salt):[3-[(4-benzoylpiperazin-1-yl)(oxo)acetyl]-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl]methylDihydrogen Phosphate, 2-amino-2-(hydroxymethyl)propane-1,3-diol Salt(1:1)

The sequence of reactions is described in Scheme for Example 10.

Scheme for Example 10

Preparation of di-tert-butyl Chloromethyl Phosphate

A mixture of tetrabutylammonium di-tert-butyl phosphate (57 g, 0.126mol, Digital Specialty Chemicals) and chloroiodomethane (221 g, 1.26mol) was stirred at room temperature for four hours before the volatileswere removed under vacuum. 500 ml of ethyl ether was added to theresidue and insoluble solid was filtered away. Concentration of thefiltrate in vacuo and removal of remaining volatiles using a vacuum pumpprovided di-tert-butyl chloromethyl phosphate as a light brown or yellowoil, which was utilized in the next step without further purification.

Preparation of IIc:(3-(2-(4-benzoylpiperazin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methyldi-tert-butyl Phosphate

NaH (2.6 g, 10.3 mmol, 95% in oil, Seq.) was added slowly into asuspension of IVc (10.0 g, 21.1 mmol) in dry THF (100 ml) and themixture was allowed to stir for 0.5 hour at room temperature. A solutionof iodine (5.27 g, 20.8 mmol) dissolved in dry THF (10 ml) was addedslowly into the stirring solution at a rate which prevented foaming or aviolent reaction. The resultant mixture was stirred for an additional 3hours before a second 2.6 g portion of NaH was introduced. After 15minutes at ambient temperature di-tert-butyl chloromethyl phosphate, theentire batch of di-tert-butyl chloromethyl phosphate, obtained from stepone, was added. After stirring for 16 hours, the reaction mixture waspoured into iced NH₄OAc (30%) (120 ml), followed by extraction withEtOAc (3×300 ml). The combined organic extracts were washed with water(100 ml) and then brine (100 ml), dried over Na₂SO₄, and concentratedunder vacuum to afford a residue, which was purified by silica gelchromatography (elution with EtOAc/Et₃N (50/1) and then EtOAc/MeOH(100/1)) to give 8.0 g (˜75% AP, ˜41% yield) of diester IIc as a lightyellow solid.

¹H NMR (500 MHz, CD₃OD) δ8.82 (s, 1H), 8.41 (s, 1H), 8.04 (s, 1H), 7.47(b, 5H), 6.00 (d, 2H, J=14.5 Hz), 4.10 (s, 3H), 4.00-3.40 (b, 8H), 2.49(s, 3H), 1.28 (s, 18H); ¹³C NMR (125 MHz, CD₃OD) δ18.6, 176.4, 172.9,168.0, 162.6, 152.6, 147.5, 144.0, 136.5, 131.5, 130.8, 129.9, 129.1,128.3, 126.1, 124.0, 116.2, 85.8, 75.4, 61.6, 57.7, 30.1, 22.2, 13.7;HRMS m/z: (M+H)⁺ calcd for C₃₃H₄₃N₇O₈P 696.29, found 696.34.

Preparation of Icb (Mono L Tromethamine Salt):[3-[(4-benzoylpiperazin-1-yl)(oxo)acetyl]-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl]methylDihydrogen Phosphate, 2-amino-2-(hydroxymethyl)propane-1,3-diol Salt(1:1)

500 mg (˜75 AP, 0.54 mmol) of diester IIc was dissolved in a mixture ofwater (2.5 ml) and acetone (2.5 ml). The resulting mixture was stirredat 40° C. for 16 hours to complete the solvolysis. To this reactionmixture was added 3.0M aqueous TRIS (mono tromethamine) solution toadjust pH to 3.32. Acetone (30 ml) was slowly added to the reactionmixture in 1 hour.* After complete addition of acetone, the solution wasstirred overnight to complete the crystallization of Icb. The solid wascollected by filtration and rinsed with 20:1 acetone-water (2×5 mL). Thewhite crystalline solid was dried under house vacuum under nitrogenatmosphere at 50° C. for 24 h to afford 290 mg of Icb (>98.5 AP). *Afteradding about 15 and 20 ml of acetone, the reaction mixture was seededwith crystalline Icb.

Icb obtained in the above operation: ¹H NMR (500 MHz, CD₃OD) δ8.83 (s,1H), 8.52 (s, 1H), 8.02 (s, 1H) 7.49 (b, 5H), 5.469 (d, 2H, J=13 Hz),4.11 (s, 3H), 4.00-3.40 (m, 8H), 3.66 (s, 6H), 2.50 (s, 3H); ¹³C NMR(125 MHz, CD₃OD) δ185.6, 171.9, 167.4, 161.4, 151.7, 146.9, 143.8,135.4, 130.3, 129.7, 128.8, 127.2, 124.9, 122.6, 114.3, 73.5, 61.8,59.9, 56.5, 46.0, 41.7, 12.6. HRMS m/z: (M-trisamine+H)⁺ calcd forC₂₅H₂₇N₇O₈P 584.1659, found 584.1664. Anal. Calcd. C, 49.43; H, 5.29; N,15.90; P, 4.39; found: C, 49.18; H, 5.38; N, 15.59; P, 4.26. MeltingPoint 203° C.

Obtained via other process (hydrolysis with TFA in methylene chloride),salt Icb is ˜1 molar mono tromethamine salt with 0.47% of water, 0.1% ofacetone and 0.05% of methanol. ¹H NMR (500 MHz, d₆-DMSO, 30° C.) δ8.77(s, 1H), 8.48 (s, 1H), 8.00 (s, 1H) 7.44 (b, 5H), 5.42 (d, 2H, J=15 Hz),4.02 (s, 3H), 3.70-3.30 (m, 8H), 3.41 (s, 6H), 2.38 (s, 3H); ¹³C NMR(125 MHz, CDCl₃, 30° C.) δ184.8, 169.0, 165.8, 160.3, 150.4, 146.2,143.2, 135.4, 129.4, 128.9, 128.2, 127.7, 126.9, 123.2, 122.2, 112.9,72.3, 60.7, 59.0, 56.7, 13.4. MS m/z: (M-trisamine+H)⁺ calcd forC₂₅H₂₇N₇O₈P 584.2, found 584.0. Anal. Calcd. C, 49.11; H, 5.37; N,15.76; P, 4.32; found: C, 48.88; H, 5.28; N, 15.71; P, 4.16. M.P.201-205° C.

General Procedure to Form Additional Salts of Iac

Procedure A:

1.2 eq. of metal alkoxide was added into a solution of phosphoric acidin THF and precipitate was collected as salt form.

Procedure B:

2.2 eq. (for Na, K) or 1.2 eq. (for Mg) of metal alkoxide was added intoa solution of phosphoric acid in THF. After 2 hours, solvent wasevaporated and MeOH was added to provide a clear solution. EtOH or iPrOHwas then added into the solution until it became cloudy. Then, MeOH wasadded cautiously to let solution become just clear again. The mixedsolution was left open to air for 16 hours and resultant precipitate wascollected as the salt form.

Procedure C:

2.2 eq. of amine was added into a solution of phosphoric acid in THF.After 2 hours, solvent was evaporated and MeOH was added to provide aclear solution. EtOH or iPrOH was then added into the solution until itbecame cloudy. Then, MeOH was added cautiously to let solution becomejust clear again. The mixed solution was left open to air for 16 hoursand resultant precipitate was collected as salt form.

Element Analysis Element Analysis (Theoretical (Observed Comp. ProcedureCompound Parent Molecule Comp.) %) Used Iac

C 51.88% H 4.73% N 10.52% P 5.82% Element Analysis Element Analysis(Theoretical (Observed Comp. Salt Form Comp.) %) Mix of Iad with smallamount of Ia

Mono Na salt C 49.83% H 4.36% N 10.11% P 5.59% Na 4.15% Di Na salt C47.93% H 4.02% N 9.72% P 5.37% Na 7.89% C 47.43% H 4.59% N 9.24% P 5.72%Na 4.48% Mainly mono Na salt Procedure B NaOMe used Mix of Iae And Iac

Mono K salt C 48.42% H 4.24% N 9.82% P 5.43% K 6.85% Di K salt C 45.39%H 3.81% N 9.21% P 5.09% K 12.85% C 49.36% H 5.25% N 9.56% P 4.25%(4.33%) K 3.31% (3.18%) Mixture of K salt and free acid Procedure B KOMeused ~1:1 mix of Iaf and Iag

0.5 Ca salt C 50.09% H 4.39% N 10.16% P 5.62% Ca 3.63% Mono Ca salt C48.42% H 4.06% N 9.82% P 5.43% Ca 7.03% C 46.77% H 4.22% N 9.33% P 5.52%(5.70%) Ca 4.95% (5.30%) Mixture of 0.5 Ca salt and mono Ca salt (~1:1)Procedure A Ca(OMe)₂ used Iaj

0.5 Zn salt C 48.97% H 4.29% N 9.93% P 5.49% Zn 5.80% Mono Zn salt C46.36% H 3.89% N 9.40% P 5.20% Zn 10.97% C 44.87% H 4.08% N 9.00% P5.54% (5.45%) Zn 9.18% (9.47%) Mainly mono Zn salt Procedure AZn(O—tBu)₂ used Mainly Iak

0.5 Mg salt C 50.82% H 4.45% N 10.31% P 5.70% Mg 2.24% Mono Mg salt C49.80% H 4.18% N 10.10% P 5.58% Mg 4.38% C 46.22% H 4.48% N 8.90% P3.88% Mg 3.60% Mainly mono Mg salt Procedure B Mg(OEt)₂ used Mix of Iamand Ian

Mono Tris salt C 49.62% H 5.55% N 10.72% P 4.74% Di Tris salt C 48.06% H6.11% N 10.85% P 4.00% C 45.30% H 5.60% N 9.59% P 2.98% Mainly di Trissalt mixed with Tris Procedure C Tromethamine used

Example 11 Preparation of Compound II′a from IIa

Di-phosphate ester IIa (500 mg) was dissolved in 5 ml of 10% TFA in THF.The reaction mixture was stirred at room temperature for 4 hours beforebeing quenched by 10% aqueous Na₂CO₃ solution (30 ml). After beingwashed with EtOAc (50 ml), the aqueous phase was concentrated undervacuum to provide a residue which was purified using Shimadzu automatedpreparative HPLC System to afford the desired mono-phosphate II′a (26.5mg)). ¹H NMR (500 MHz, CDCl₃) d 8.13 (s, 1H), 7.40 (b, 6H), 6.13 (d, 2H,J=11.5 Hz), 4.05 (s, 3H), 3.88 (s, 3H), 3.90-3.40 (m, 8H), 1.39 (s, 9H);MS m/z: (M+H)⁺ calcd for C27H34N4O9P 589.21, found 589.13; LC retentiontime 1.32 min (column: Xterra 4.6×50 mmC18 5 um).

Alternate Preparation of di-tert-butyl Chloromethyl Phosphate

Reaction

To the inerted reactor, di-tert-butyl potassium phosphate (1.69 kg), 4.0eq. of sodium carbonate and 0.05 eq of tetrabutyl ammonium hydrogensulfate were added through the manway. Methylene chloride (7.7 L/kg) wasthen pumped through the sprayball to wash down the reactor walls. Withthe jacket temperature below 10° C., the exothermic water charge wasadded over the course of ten minutes (7.6 L/kg). The associated exothermwas minor with a batch temperature rising from 11.1 to 16.2° C. over thecourse of the addition. With the jacket and batch temperature near 7 and15° C., respectively, 2.0 eq. of chloromethylsulfonylchloride (CMCS) wascharged via addition funnel. The charge continued for 2 hours while thejacket temperature was slowly raised to 20° C. during the charge. Themaximum batch temperature during the CMCS charge was 25.3° C. The jackettemperature was slowly raised during the charge to ensure that theexotherm started as preliminary laboratory data indicated that theexotherm may be slowed at lower batch temperatures. The reaction mixturewas agitated and after 3.5 hours, an NMR sample indicated that thereaction had progressed 72%. The reaction was allowed to proceedovernight with a batch temperature between 19.7 and 23.6° C. (Delta Vhistorian). An NMR sample taken after 16 hours indicated a reactionconversion of 76%. Laboratory batches ranged in conversion from 60 to80%.

Work-Up

After the reaction was deemed complete, additional water at 9.3 L/kg wasadded to the batch to affect a phase split. The product rich lower phasewas transferred to a carboy and the upper aqueous phase was sent towaste. A small rag layer was kept with the product rich organic. Theorganic phase was returned to R-1A and additional water at 5.1 L/kg wasadded as a wash. The phases were split with the product rich organic,approximately 18.5 kg, being sent to a carboy while the upper aqueousphase was sent to waste. No rag layer or solids were observed in thesecond split. However, it is recommended to polish filter the productrich organic to remove precipitated salts.

Methylene Chloride Distillation

The product rich organic was transferred to the rotovap bowl ofEVAPO-1A. Distillation of the methylene chloride was initiated with ajacket temperature of approximately 22° C. The distillation rate slowedafter 4.5 hours and a batch sample was taken to analyze the methylenechloride content. NMR analysis indicated a 4:1 ratio of di-tert-butylchloromethyl phosphate to methylene chloride. Typical laboratory resultsof this stream would indicate a 10:1 ratio so the distillation wascontinued with an increase in the rotovap jacket temperature. After anadditional 2.5 hours, the distillation rate stopped. An NMR sample ofthe batch indicated that the ratio had increased to 5:1 di-tert-butylchloromethyl phosphate to methylene chloride. The maximum rotovap jackettemperature was 28.4° C.

Purity of the di-tert-butyl Chloromethyl Phosphate Oil

NMR analysis of the di-tert-butyl chloromethyl phosphate oil indicatedthe potency to be greater than 100%. Development work typically producedmaterial with a potency of 100±10%. Karl-Fischer analysis measured watercontent at 0.02 wt % and GC analysis measured methylene chloride at10.69 wt %. Thus, the reported potency is 89.29 wt % accounting for themethylene chloride and water contribution in the oil.

Storage of di-tert-butyl Chloromethyl Phosphate

The di-tert-butyl chloromethyl phosphate oil was placed in cold room andthe temperature was monitored with a stripchart recorder. Laboratorybatches were typically held between 0 to 5° C. An NMR of the productafter the 104 hour hold in the cold room indicated that the material hadnot lost potency.

(Safety testing conducted during the campaign indicates that uponholding the oil self-heats with a subsequent pressure build-up).

NMR Standard Prep and Sample Prep

Preparation of Trimethyl Phosphate (TMPO₄) Standard Solution

A standard solution of TMPO₄ should be prepared based on a 100 M %theoretical yield. For example: a 10 g input of the di-t-butyl potassiumphosphate salt should yield 10.41 g (0.402 mols) of di-tert-butylchloromethyl phosphate. The volume of dichloromethane in the reactionmixture will be 75 mL. The molarity of the solution is 0.536. A TMPO₄solution should be prepared in that molarity and 0.5 mL of that solutionshould be combined with 0.5 mL of the dichloromethane layer from thereaction. The integrals found in the ³¹P NMR can be directly comparedand will give the % conversion of di-tert-butyl chloromethyl phosphate.

Determination of % Unreacted Starting Material in the Reaction AqueousPhase:

After recording the volume of the reaction aqueous phase, accuratelytransfer 0.500 mL into a 1-dram vial containing a known weight ofinternal standard TMPO₄. Add approximately 0.24 mL of D₂O. Shake to mixthoroughly. Obtain ³¹P-quant spectra.

Calculation % Unreacted Starting Material:

${\frac{{sp}.\mspace{14mu}{aq}.\mspace{14mu}{vol}.}{G\mspace{11mu}{{act}.\mspace{11mu}{inpt}.}} \times \frac{{mg}\mspace{14mu}{TMPO}_{4}}{{{vol}.\mspace{14mu}{sample}}\mspace{14mu}\left( {0.5\mspace{14mu}{mL}} \right)} \times \frac{248.30\mspace{14mu}{MW}\mspace{14mu}{{st}.\mspace{14mu}{mat}.}}{140.08\mspace{14mu}{MW}\mspace{14mu}{TMPO}_{4}} \times \frac{{\,^{31}P}\mspace{14mu}{NMR}\mspace{14mu}{integration}\mspace{14mu}{{st}.\mspace{14mu}{mat}.}}{{\,^{31}P}\mspace{14mu}{NMR}\mspace{14mu}{integration}\mspace{14mu}{TMPO}_{4}} \times \frac{100}{1000}} = {\%\mspace{14mu}{unreacted}\mspace{14mu}{starting}\mspace{14mu}{material}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{{rxn}.\mspace{14mu}{aq}.}}$

Example

${{\frac{212\mspace{14mu}{mL}}{11.0\mspace{14mu} g\mspace{14mu}{{act}.}} \times \frac{13.4\mspace{14mu}{mg}\mspace{14mu}{TMPO}_{4}}{0.500\mspace{14mu}{mL}} \times \frac{248.30\mspace{14mu}{MW}\mspace{14mu}{{st}.\mspace{14mu}{mat}.}}{140.08\mspace{14mu}{MW}\mspace{14mu}{TMPO}_{4}} \times \frac{9.617}{270.392} \times \frac{100}{1000}} = {3.26\%\mspace{14mu}{unreacted}\mspace{14mu}{starting}\mspace{14mu}{material}}}\;$Determination of the %-Potency of the Product Oil:

After recording the net weight of the distilled product oil, tare a1-dram, screw-top vial containing a known weight of internal standardTMPO₄. Transfer approximately 0.02 mL of product oil into the vial andrecord the net weight of product oil. Add approximately 0.7 mL of CDCl₃.Shake to mix thoroughly. Obtain ³¹P-quant spectra. Inspect the ¹H NMRspectra for the presence of residual phase-transfer catalyst(tetra-n-butyl-ammonium bisulfate) and methylene chloride. Report theseas mol % relative to product.

Calculation of %-Potency:

${\frac{{mg}\mspace{14mu}{TMPO}_{4}}{{mg}\mspace{14mu}{sample}} \times \frac{258.68\mspace{20mu}{MW}\mspace{11mu}{product}}{140.08\mspace{20mu}{MW}\mspace{14mu}{TMPO}_{4}} \times \frac{{\,^{31}P}\mspace{14mu}{NMR}\mspace{14mu}{integration}\mspace{14mu}{product}}{{\,^{31}P}\mspace{14mu}{NMR}\mspace{14mu}{integration}\mspace{14mu}{TMPO}_{4}} \times 100} = {\%\mspace{14mu}{potency}\mspace{14mu}\left( {w/w} \right)\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{product}\mspace{14mu}{oil}}$

Example

${\frac{13.7\mspace{14mu}{mg}\mspace{14mu}{TMPO}_{4}}{22.3\mspace{14mu}{mg}\mspace{14mu}{sample}} \times \frac{258.68\mspace{20mu}{MW}\mspace{14mu}{product}}{140.08\mspace{14mu}{MW}\mspace{14mu}{TMPO}_{4}} \times \frac{44.744}{55.256} \times 100} = {91.9\;\%\mspace{14mu}{potency}\mspace{14mu}\left( {w/w} \right)\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{product}\mspace{14mu}{oil}}$

NMR Data for di-tert-butyl Chloromethyl Phosphate

¹H NMR (300.13 MHz, CDCl₃): δ 1.52 (s, 18H), δ 5.67 (d, J=15.5, 2H) ¹³CNMR (75.47 MHz, CDCl₃): δ 29.77 (d, J=4.5, 6C), δ 73.34 (d, J=7.5, 1C),δ 84.16 (d, J=1.5, 2C)

³¹P NMR (121.49 MHz, CDCl₃): δ −10.51 (s, 1P)

Example 12 Alternate Preparation of Iab (Pro-Drug of IVa)

A 500 ml 4-neck round bottom flask equipped with an overhead stirrer,thermocouple, addition funnel, a nitrogen inlet and a septa was chargedIVa (20.01 g, 47.37 mmol), K₂CO₃ (13.13 g, 95.00 mmol) and DMSO (100 ml,1.41 moles) and the reaction was stirred at room temperature resultingin a light brown heterogeneous suspension. Di-tert-butyl chloromethylphosphate (14.83 g, 57.32 mmol) was added via addition funnel and thereaction was heated to 30° C. for 16-24 hours after which time thereaction was cooled to 10° C. To the reaction was added DCM (200 ml)then was slowly quenched with water (200 ml) maintaining the reactiontemperature under 20° C. resulting in a biphasic mixture. The productrich bottom layer was separated, washed with water (200 ml), thentransferred to a 500 ml 4-neck round bottom flask equipped with anoverhead stirrer, thermocouple, addition funnel, and nitrogen inlet.Trifluoroacetic acid (53.0 ml, 700.94 mmol) was added via additionfunnel resulting in a slight exotherm. The reaction was stirred for 1-3hours then cooled to 0° C. Methanol (300 ml) was added keeping thereaction temperature under 20° C., and then the cooled to 0° C. Thereaction flask was fitted with a distillation apparatus and concentratedunder vacuum to a volume of 200 ml (200 torr, <30° C.). The reaction wasseeded with Iac (0.200 g) then stirred overnight at room temperatureresulting in a slurry. The slurry was filtered then the wet cake waswashed with THF (300 ml) then dried in a vacuum oven at 50° C. overnightresulting in a pale yellow to white powder (23.94 g, 95%). ¹H NMR (400MHz, DMSO-d6) δ 8.30 (s, 1H), 7.55 (s, 1H), 7.44 (s, 5H), 6.12 (d,J=10.6 Hz, 2H), 3.97 (s, 3H), 3.85 (s, 3H), 3.80-3.22 (m, 8H); ¹³C NMR(100 MHz, DMSO-d6) δ 185.49, 169.26, 166.06, 146.20, 145.60, 140.64,135.50, 129.68, 128.41, 127.04, 123.46, 121.17, 120.08, 114.32, 72.43,56.92, 53.32, 45.22, 40.50; ES⁺ MS m/z (rel. intensity) 533 (MH⁺, 100),453 (MH⁺−H3PO4, 15).

To a 10 L 4 neck reactor equipped with a thermocouple, overhead stirrer,condenser and nitrogen inlet was added Iac (611 g, 1.15 mol) and water(3875 ml). To the resulting suspension was added lysine (168 g, 1.15mol). The reaction was stirred for one hour at RT, heated to 50° C. thenmaintained at 50° C. with stirring for an additional hour. The resultinghazy solution (pH=4.55) was filtered through a 10 micron cuno filterinto a 20 L 4 neck reactor equipped with a thermocouple, overheadstirrer, condenser and nitrogen inlet. The reaction was heated to 50° C.then acetone (8 L) was added rapidly. The reaction was allowed to warmto 50° C. then acetone (4 L) was added at a moderate rate keeping thereaction temperature above 45° C. The reaction was seeded with Iab(0.200 g) then cooled to room temperature over 5 hours resulting in aslurry. The slurry was stirred overnight at room temperature thenfiltered. The wet cake was washed with acetone (4 L) then dried in avacuum oven at 25° C. overnight with a bleed of moist air resulting in afluffy white powder (751 g, 96%).

Example 13 Alternate Preparation of Icb (Pro-Drug of IVc)

To a 10 L reactor equipped with an overhead stirrer, thermocouple,distillation apparatus, and nitrogen inlet was charged IVc (200.00 g,422.39 mmol), Cs₂CO₃ (344.06 g, 1.06 mol), KI (140.24 g, 844.81 mmol)and NMP (1.00 L, 10.38 mol). The reaction was stirred at roomtemperature resulting in a light brown heterogeneous suspension.Di-tert-butyl chloromethyl phosphate (273.16 g, 1.06 mol) was added viaaddition funnel and the reaction mixture was heated to 30° C. for 16-24hours with stirring after which time the reaction was cooled to 5° C. Tothe reaction was added DCM (1.5 L) then the reaction was slowly quenchedwith water (3.5 L) maintaining the reaction temperature under 20° C.resulting in a biphasic mixture. The product rich bottom layer wasseparated, washed with water (3.5 L×3), then transferred back to thereactor. The solution was concentrated under vacuum to a volume of 1 Lkeeping the temperature below 25° C. IPA was added (2 L) then thereaction was concentrated under vacuum to a volume of 2 L keeping thetemperature below 25° C. The reaction was then seeded with IIc (0.200g), stirred overnight at room temperature resulting in a slurry. Theslurry was filtered and the wet cake was washed with MTBE (1 L), driedin a vacuum oven at 50° C. overnight resulting in a yellow/white powder(207.1 g, 70%). ¹H NMR (400 MHz, CDCl₃) δ 8.54 (s, 1H), 8.18 (s, 1H),7.91 (s, 1H), 7.42 (s, 5H), 5.95 (d, J=14.2 Hz, 2H), 4.06 (s, 3H),3.97-3.36 (m, 8H), 2.50 (s, 3H), 1.27 (s, 18H); ¹³C NMR (100 MHz, CDCl₃)δ 184.64, 170.65, 165.91, 161.60, 150.82, 145.38, 141.89, 134.96,130.20, 129.59, 128.68, 127.58, 127.10, 124.77, 122.64, 115.22, 83.90,83.83, 73.69, 73.63, 56.95, 46.04, 41.66, 29.61, 29.56, 13.90; ES⁺ MSm/z (rel. intensity) 696 (MH⁺, 10), 640 (MH⁺−isobutylene, 30), 584(MH⁺−2 isobutylene, 100).

To a 10 L 4 neck reactor equipped with a thermocouple, overhead stirrer,condenser and nitrogen inlet was added IIc (200.24 g, 287.82 mmol),acetone (800.00 ml, 10.88 mol) and water (800.00 ml, 44.41 mol). Thereaction was heated to 40° C. and stirred for 18-24 hours. The reactionwas cooled to 20° C. then tromethamine (33.62 g, 277.54 mmol) was added.The reaction was heated to 40° C. then stirred for an additional houruntil all solids were dissolved. The reaction was cooled to 20° C. thenfiltered through a 10 micron cuno filter into a 10 L 4 neck reactorequipped with a thermocouple, overhead stirrer, and nitrogen inlet.Acetone (3 L) was added rapidly, followed by seeding with Icb (0.500 g),then additional acetone (3 L) was added. The reaction was stirred atroom temperature overnight resulting in a slurry then filtered. The wetcake was washed with acetone (800 ml) then dried in a vacuum oven at 50°C. overnight resulting in a fluffy white powder (165.91 g, 82%).

Supplementary Information:

Isolation of the Free-Acid Intermediate Ic:

In a 250 mL 3 neck reactor equipped with a thermocouple, overheadstirrer, condenser and nitrogen inlet was added IIc (10.0 g, 14.37mmol), acetone (40.00 ml, 544.15 mmol) and water (40.00 ml, 2.22 mol).The reaction was heated to 40° C. and stirred for 14-24 hours. Thereaction was cooled to 20° C. then stirred for three hours, resulting ina slurry. The slurry was filtered, then the wet cake washed with acetone(40.00 ml) then dried in a vacuum oven at 50° C. overnight resulting ina fluffy white powder (7.00 g, 83%). NMR (400 MHz, DMSO-d6) δ 8.84 (s,1H), 8.47 (s, 1H), 8.06 (s, 1H), 7.45 (s, 5H), 5.81 (d, J=12.3 Hz, 2H),4.03 (s, 3H), 3.91-3.19 (m, 8H), 2.39 (s, 3H); ¹³C NMR (500 MHz,DMSO-d6) δ 185.20, 169.32, 165.85, 160.75, 150.51, 146.30, 143.24,135.53, 129.74, 129.22, 128.46, 127.34, 127.09, 123.67, 122.73, 113.94,72.90 (d, ²J_(C-P)=5 Hz), 57.01, 45.2 (bs), 40.8 (bs), 13.66. ES⁺ MS m/z(rel. intensity) 486 (MH⁺−H₃PO₄, 100).

Example 14 Alternate Preparation of Ibb (Pro-Drug of IVb)

To a 10 L reactor equipped with an overhead stirrer, thermocouple, andnitrogen inlet was charged IVb (400.00 g, 894.73 mmol), Cs₂CO₃ (873.70g, 2.68 mol), KI (297.70 g, 1.79 mol) and NMP (1.00 L, 10.38 mol). Thereaction mixture was stirred at room temperature resulting in a lightbrown heterogeneous suspension. Di-tert-butyl chloromethyl phosphate(460.50 g, 1.78 mol) was added via addition funnel and the reaction washeated to 30° C. for 16-24 hours at which time the reaction was cooledto 5° C. To the reaction was added n-BuOAc (2.4 L) then the reaction wasslowly quenched with water (4 L) maintaining the reaction temperatureunder 20° C. resulting in a biphasic mixture. The bottom aqueous layerwas removed from the reactor, then the product rich top layer was seededwith IIc, (0.40 g) then stirred 3 hours at room temperature resulting ina slurry. The slurry was filtered then the wet cake was washed with MTBE(1.6 L) then dried in a vacuum oven at 50° C. overnight resulting in ayellow/white powder (483.2 g, 81%). ¹H NMR (400 MHz, CDCl₃) δ 8.35 (s,1H), 8.27 (s, 1H), 8.22 (s, 1H), 7.90 (s, 1H), 7.42, (s, 5H), 5.92, (d,J=14.9 Hz, 2H), 4.02-3.40 (m, 8H), 1.24 (s, 18H); ¹³C NMR (100 MHz,CDCl₃) δ 182.75, 170.64, 152.07, 144.03, 134.91, 133.96, 131.82, 130.21,128.68, 128.27, 128.00, 127.07, 125.81, 124.01, 113.82, 84.00, 83.93,73.97, 46.12, 41.90, 29.57, 29.53; ES⁺ MS m/z (rel. intensity) 558 (MH⁺,100).

To a 300 ml 4 neck reactor equipped with a thermocouple, overheadstirrer, condenser, and nitrogen inlet was added Ith (18.0 g, 26.87mmol), IPA (36.00 ml, 470.92 mol) and water (36.00 ml, 2.0 mol). Thereaction was heated to 40° C. and stirred for 18-24 hours. The reactionwas cooled to 20° C. then lysine (3.73, 25.54 mmol) was added. Thereaction was stirred for 1 hour until all solids were dissolved. IPA (54ml) was added over 30 minutes followed by seeding with Ibb (0.180 g) andstirring for an additional 30 minutes. IPA was added (18 ml) over 1 hourthen the reaction was heated to 50° C. resulting in a thin slurry. Thereaction was seeded with Ibb (0.180 g) then IPA (36 ml) was added over 2hours then stirred for 12 hours resulting in a slurry. The reaction washeated to 70-80° C. for 2 to 3 hours then cooled to 50° C. IPA (59 ml)was added over 1 hour then additional IPA (121 ml) was added over 1hour. The reaction was cooled to 20° C. over 2 hours then stirred for anadditional 2 hours then filtered. The wet cake was washed with IPA (180ml) then dried in a vacuum oven at 50° C. overnight resulting in a whitepowder (15.43 g, 82%).

Supplementary Information:

Procedure for the Isolation of the Free-Acid Intermediate Ibc:

In a 500 ml 3 neck flask equipped with a thermocouple, overhead stirrer,condenser, and nitrogen inlet was added IIb (50.00 g, 74.76 mmol),Acetone (100.00 ml, 1.36 mol) and water (100.00 ml, 5.55 mol). Thereaction was heated to 40° C. and stirred for 18-24 hours.

In a 250 ml 3 neck flask equipped with a pH probe, magnetic stirbar, andnitrogen inlet was added 150 ml of the above Ibc solution then the pHwas adjusted to pH=6.2 with 10 N NaOH. The solution was transferred to aseparatory funnel, then washed with EtOAc (100 ml) then DCM (100 ml),then transferred back the 250 ml 3 neck flask. The pH was adjusted topH=1.3 with 2 N HCl followed by stirring for three hours, resulting in aslurry which was filtered. The wet cake was re-slurried in MTBE (150 ml)then filtered, followed by re-slurrying in THF/Water (100:1, 130 ml) for45 minutes, then filtered and dried in a vacuum oven at 50° C. overnightresulting in a white powder (10.0 g, 33%). NMR (400 MHz, DMSO-d6) δ 8.69(s, 1H), 8.62 (s, 1H), 8.42 (s, 1H), 7.98 (s, 1H), 7.41 (s, 5H), 5.47(d, J=13.3 Hz, 2H), 3.99-3.18 (m, 8H); ¹³C NMR (100 MHz, DMSO-d6) δ183.97, 169.23, 165.20, 151.69, 145.91, 135.48, 133.83, 131.59, 129.65,129.11, 129.03, 128.42, 127.77, 127.49, 127.03, 122.62, 112.08, 72.57.ES⁺ MS m/z (rel. intensity) 558 (MH⁺, 100).

Example 15 Preparation of Prodrug Ie

Step One

Preparation of2-(1-(2-(4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoacetyl)piperidin-4-ylidene)-2-(pyridin-2-yl)acetonitrile(IVe)

2-(4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoaceticacid (1.5 g), 2-(piperidin-4-ylidene)-2-(pyridin-2-yl)acetonitrilehydrochloride (1.5 g),3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) (2.1 g)and Hunig's Base (2 ml) were combined in 20 ml of DMF. The mixture wasstirred at room temperature for 16 hours. DMF was removed viaevaporation at reduced pressure and the residue was partitioned withMeOH (80 ml). The precipitate was collected via filtration to provide0.85 g of the product,2-(1-(2-(4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pynolo[2,3-c]pyridin-3-yl)-2-oxoacetyl)piperidin-4-ylidene)-2-(pyridin-2-yl)acetonitrile(IVe). ¹H NMR (500 MHz, DMSO-d6) δ12.42 (s, 1H), 9.23 (m, 1H), 8.69 (m,1H), 8.27 (m, 1H), 7.89 (m, 2H), 7.58 (m, 1H), 7.52 (m, 1H), 3.98 (s,3H), 3.99-2.70 (m, 8H), 2.60 (m, 3H). MS m/z: (M+H)⁺ calcd forC₂₅H₂₃N₈O₃ 483.19, found 483.18.

Step Two

Phosphate ester (45.1 g, 0.1 mol) and chloroiodomethane (200 g, 1.14mol) were combined in 100 ml of benzene and the mixture was stirred atroom temperature for four hours before benzene was removed under vacuum.Then, 500 ml of ethyl ether was added to the residue and insoluble solidwas filtered away. Concentration of the filtrate provided di-tert-butylchloromethyl phosphate, which was utilized in the next step without anyfurther purification.

Step Three

NaH (0.2 g, 95%) was added slowly into a suspension of2-(1-(2-(4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoacetyl)piperidin-4-ylidene)-2-(pyridin-2-yl)acetonitrile(IVe) in dry THF (20 ml) and the mixture was allowed to stir for an hourat room temperature. Iodine (0.4 g) dissolved in dry THF (2 ml) wasadded slowly into the stirring solution. The mixture was stirred foradditional 3 hours before 0.2 g of NaH was charged. Following completionof addition the resultant mixture was stirred at ambient temperature foran additional 15 minutes and then di-tert-butyl chloromethyl phosphate,obtained from step two, was added. After stirring for 16 hours, thereaction mixture was poured into iced NH₄OAc (30%) (50 ml), followed byextraction with EtOAc (3×100 ml). The combined organic extracts werewashed with water (50 ml) and then brine (50 ml), dried over Na₂SO₄, andconcentrated under vacuum to afford a residue, which was purified bysilica gel chromatography (elution with EtOAc/Et₃N (100/1)) to give 330mg of di-tert-butyl(3-(2-(4-(cyano(pyridin-2-yl)methylene)piperidin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methylphosphate (IIe). ¹H NMR (500 MHz, CD₃OD) δ38.85 (m, 1H), 8.66 (m, 1H),8.45 (m, 1H), 8.06 (m, 1H), 7.92 (m, 1H), 7.60 (m, 1H), 7.43 (m, 1H),6.05 (m, 2H), 4.11 (s, 3H), 4.00 (m, 1H), 3.82 (m, 1H), 3.76 (m, 1H),3.60 (m, 1H), 3.04 (m, 1H), 2.95 (m, 1H), 2.85 (m, 1H), 2.80 (m, 1H),2.52 (s, 3H), 1.30 (m, 18H). MS m/z: (M+H)⁺ calcd for C₃₄H₄₂N₈O₇P705.29, found 605.30.

Step Four

Di-tert-butyl(3-(2-(4-(cyano(pyridin-2-yl)methylene)piperidin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methylphosphate (IIe) was dissolved in 8 ml of a mixed solution of TFA anddichloromethane (10% TFA/CH2Cl2) and the mixture was stirred for threehours. All the solvents were removed under vacuum and the residue waspurified using a Shimadzu automated preparative HPLC System to give 25mg of tert-butyl(3-(2-(4-(cyano(pyridin-2-yl)methylene)piperidin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methylhydrogen phosphate (II′e) and 33 mg of(3-(2-(4-(cyano(pyridin-2-yl)methylene)piperidin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methyldihydrogen phosphate (Ie).

tert-Butyl(3-(2-(4-(cyano(pyridin-2-yl)methylene)piperidin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methylhydrogen phosphate (II′e): ¹H NMR (500 MHz, CD₃OD) δ38.83 (m, 1H), 8.55(m, 1H), 8.35 (m, 1H), 7.92 (m, 2H), 7.54 (m, 1H), 7.41 (m, 1H), 5.86(m, 2H), 3.98 (s, 3H), 3.96 (m, 1H), 3.72 (m, 1H), 3.65 (m, 1H), 3.47(m, 1H), 2.92 (m, 1H), 2.85 (m, 1H), 2.71 (m, 1H), 2.65 (m, 1H), 2.40(s, 3H), 1.15 (m, 9H). MS m/z: (M+H)⁺ calcd for C₃₀H₃₄N₈O₇P 649.23,found 649.22.

(3-(2-(4-(Cyano(pyridin-2-yl)methylene)piperidin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methyldihydrogen phosphate (Ie): ¹H NMR (500 MHz, DMSO-d6) δ8.88 (m, 1H), 8.65(m, 1H), 8.50 (m, 1H), 8.06 (m, 1H), 7.90 (m, 1H), 7.49 (m, 2H), 5.82(m, 2H), 4.04 (s, 3H), 3.96 (m, 1H), 3.88 (m, 1H), 3.72 (m, 1H), 3.46(m, 1H), 2.94 (m, 1H), 2.82 (m, 2H), 2.73 (m, 1H), 2.40 (m, 3H); ¹³C NMR(125 MHz, DMSO-d6) δ185.2, 165.6, 160.6, 159.1, 151.0, 150.4, 149.5,146.2, 143.1, 137.4, 129.1, 127.2, 124.4, 123.6, 122.6, 117.4, 116.3,113.9, 110.0, 72.8, 56.9, 48.4, 44.4, 36.4, 34.0, 13.6. MS m/z: (M+H)⁺calcd for C₂₆H₂₆N₈O₇P 593.17, found 593.14.

Example 16 Preparation of Prodrug If

To a mixture of IVf (99.5 mg, 0.21 mmol) in 1-methyl-2-pyrrolidinone(1.0 ml) at r.t. in a capped vial was added KI (144 mg, 0.87 mmol) andCs₂CO₃ (416 mg, 1.28 mmol)), and the mixture was stirred for about 5min. Di-tertbutyl chloromethyl phosphate reagent (218 mg, 0.84 mmol) wasthen added dropwise. The resulting mixture was then stirred at 35 to 40°C. for 20 hours. The mixture was then diluted with H₂O (about 8 ml) andextracted with EtOAc (about 8 ml). The organic extract was separated andevaporated to give the di-t-butyl chloromethyl phosphate; AnalyticalHPLC method: Solvent A 10% MeOH—90% H₂O—0.1% TFA; Solvent B 90% MeOH—10%H₂O—0.1% TFA; Start % B=0, Final % B=100, Gradient time=2 min, FlowRate=5 mL/min, Column: Xterra MS C18 S7 3.0×50 mm; LC/MS: (ES) m/z(M+H)⁺=694.22, HPLC R_(t)=1.243.

A mixture of Intermediate IIf in H₂O/isopropanol (1.0 ml/1.0 ml) in astoppered round bottom flask was stirred at 40° C. for 9.5 hours. Themixture was then cooled to r.t., and the solution transferred to a vialby using a pipette. MeCN (1.0 ml) was added to this solution, which wasthen added isopropanol slowly and with intermittent stirring using aspatula. The off white precipitates were then filtered, washed withisopropanol (2×1.0 ml) and then dried under high vacuum to give theprodrug If; ¹HNMR (500 MHz): (DMSO-d₆) δ 8.76 (s, 1H), 8.71 (s, 1H),8.69 (s, 1H), 8.49 (s, 1H), 8.09 (d, J=8, 1H), 8.06 (s, 1H), 7.89 (d,J=7, 1H), 7.85 (app t, 1H), 7.59 (app t, 1H), 5.68 (d, J=13, 2H), 4.00(b m, 2H), 3.90 (b m, 2H), 3.85 (b m, 2H), 3.65 (b m, 2H); AnalyticalHPLC method: Solvent A 10% MeOH—90% H₂O—0.1% TFA; Solvent B 90% MeOH—10%H₂O—0.1% TFA; Start % B=0, Final % B=100, Gradient time=2 min, FlowRate=5 mL/min, Column: Xterra MS C18 S7 3.0×50 mm; LC/MS: (ES) m/z(M+H)⁺=582.00, HPLC R_(t)=1.797.

To be successful, the conversion of the prodrug into parent must beinitiated by alkaline phosphatases in man. Qualitative in vitro studiesusing human placental alkaline phosphatase and in vivo studies in ratsshowed that conversion of prodrug was rapid both in vitro with humanenzymes and in in vivo in rats. Ideally, the rate of conversion will berapid so that only limited exposure to prodrug occurs and maximumexposure to active parent antiviral agent will result. Data from studies(below) shows that in all three prodrug examples evaluated in rats, theprodrug is rapidly converted to active parent drug and that plasmalevels of prodrug are very low in comparison to parent drug at all datapoints. These studies were done at doses in which doses of parent drugand the dose equivalent from phosphate prodrug were low andapproximately equal, ˜5 mg/kg. Since the advantages of prodrugs are toovercome dissolution limited absorption, at low doses the advantages ofthe prodrugs over less soluble parent molecules for clinical use inpatients will not be obvious. The low dose in vivo studies were used todetermine if the prodrugs were generating parent molecule. Thesolubility of the parent molecules IV, is dependent on crystalline form.A crystalline form is preferred for drug development. The data for allof the parent molecules can be summarized by saying that the aqueoussolubility of crystalline material for all the parent molecules IV is<50 μg/mL and in some cases much less. Thus, the intrinsic aqueoussolubility of the parent molecules is low and plays a major role incausing dissolution-limited absorption at higher doses.

TABLE 1 Biological and Pharmaceutical Properties of N-methyl dihydrogenphosphate (or salts) Azaindoleoxoacetic Piperazine Derivatives CompoundIa Compound Ib Compound Ic (disodium salt) (disodium salt) (acid form)Solubility >18 >8 1 (mg/mL), pH 6.5 In vitro conversion Complete andrapid Complete and rapid Complete and rapid in Alkaline conversion toparent conversion to parent conversion to parent phosphatase (notewithout any without any without any intermediate 1) intermediateintermediate formation formation formation In vivo conversion Rapidgeneration of Rapid generation of Rapid generation of in Rats-oral (noteparent in plasma parent in plasma parent in plasma 2) MAP study In vivoconversion Rapid generation of Rapid generation of Rapid generation ofin Rats-iv (note parent in plasma parent in plasma parent in plasma 2)MAP study note 1: The prodrug derivative (conc. ~0.2 mM) was incubatedwith alkaline phosphatase (human placenta, Sigma, ~1.4 unit) in pH 8Tris buffer (conc. ~0.03M, 1 mL), and disappearance of the prodrug andformation of the parent were monitored by HPLC and LC/MS. In most cases,the prodrug completely disappeared, corresponding with formation of theparent within an hour or two, and no other intermediate was detected.note 2: The prodrug was administered in rats by the oral (at the doseequiv. to 5 mg/Kg of the parent) or intravenous route (at the doseequiv. to 1 mg/Kg of the parent). The plasma levels indicate rapidconversion to the parent with no detectable amount of the prodrug (po).

In the tables below the term “LLQ” means lower limit of quantitation(i.e., not detected).

TABLE 2 MAP Study A: Summary of PK after PO and IV Administration ofProdrug Ia to Rats. Comparison to Historic PO of Compound IVa in Rats IaIVa Historic IVa After prodrug After prodrug After dosing of parent PK(5 mg/kg po) dosing dosing IVa (Study A1)

Dose PO (mpk) 6.3 mpk equivalent to 5 mpk 5 mpk of IVa) Cmax (uM) LLQ1.4 ± 0.6 4.5 ± 1.5 Tmax (hr) LLQ 1.7  2 AUC 0-24 h LLQ 5.9 ± 1   14.9 ±6.2  (μM*hr) Cp @ 24 hr p.o. LLQ 35.2 (n = 1) 9.2 (n = 1) (nM) Dose IV(mpk) 1.26 mpk equivalent to 1 mpk 1 of ‘IVa) CL i.v. (ml/min/kg)   16 ±0.95 —  13 ± 4.6 Vss i.v. (L/kg) 0.095 ± 0.001 — 1.4 ± 0.4 T1/2 i.v.(hr) 0.14 ± 0.02 2.8 ± 1.5   4 ± 2.6 T1/2 p.o. (hr) 3.3 ± 2.2 1.9 ± 0.8AUC0 tot ratio after 0.534 IV administration* AUCtot ratio after 0.39 PO administration**

TABLE 3 MAP Study B: Summary of PK after PO and IV Administration ofProdrug Ib to Rats. Comparison to Historic PO of Compound IVb in RatsHistoric IVb After PK dosing of (5 g/kg Ib IVb parent IVb po) Afterprodrug dosing After prodrug dosing (Study B1)

Dose PO 7 mpk equivalent to 5 mpk 5 (mpk) of IVb) Cmax LLQ 3.9 ± 0.8 9.5± 2.8 (uM) Tmax (hr) LLQ 1.1    4.6 AUC LLQ 13.7 ± 2.6  86 ± 33 0-24 h(μM*hr) Cp @ 24 LLQ 7.5 (n = 1) 161  hr p.o. (nM) Dose IV 1.4 equivalentto 1 mpk 1 (mpk) of ‘IVa) CL i.v. 46 ± 10 — 1.6 ± 0.2 (ml/min/ kg) Vssi.v. 0.97 ± 0.47 — 0.49 ± 0.26 (L/kg) T1/2 i.v. 1.8 ± 1.2 1.5 ± 0.2 5.9± 4.9 (hr) T1/2 p.o. 2.4 ± 0.5 3.7 ± 0.9 (hr) AUC0 tot 0.10 ratio afterIV adminis- tration* AUCtot 0.14 ratio after PO adminis- tration**

TABLE 4 MAP Study C: Summary of PK after PO and IV Administration ofProdrug Ic to Rats. Comparison to Historic PO of Compound IVc in RatsHistoric IVc After dosing of PK (5 Ic IVc parent IVc mg/kg po) Afterprodrug dosing After prodrug dosing (Study C1)

Dose PO 6.5 mpk equivalent to 5 mpk of 5 (mpk) IVc) Cmax (uM) LLQ 10.2 ±2.1  13.4 ± 3.6  Tmax (hr) LLQ 0.8 ± 0.1 4 AUC 0-24 h LLQ  56 ± 7.5 110± 25  (μM*hr) Cp @ 24 hr LLQ 84.3 (n = 2) 61  p.o. (nM) Dose IV 1.3equivalent to 1 mpk of 1 (mpk) ‘IVa) CL i.v. 77.9 ± 44.3 — 1.3 ± 0.2(ml/min/kg) Vss i.v. 1.8 ± 2.6 —  0.4 ± 0.09 (L/kg) T1/2 i.v. 1.2 ± 1  3.2 ± 0.2 4.3 ± 1.1 (hr) T1/2 p.o. 2.7 ± 1   3.0 ± 0.3 (hr) AUC0 tot0.54 ratio after IV adminis- tration* AUC tot 0.43 ratio after POadminis- tration**Key for all three tables—2, 3 and 4:

${*{AUCtot}\mspace{14mu}{ratio}} = {{{\frac{{Total}\mspace{14mu}{AUC}\mspace{14mu}{of}\mspace{14mu}{parent}\mspace{14mu}{after}\mspace{11mu}{IV}\mspace{14mu}{administration}\mspace{14mu}{of}\mspace{14mu}{Prodrug}}{\mspace{14mu}\begin{matrix}{{Total}\mspace{14mu}{AUC}\mspace{14mu}{of}\mspace{14mu}{parent}\mspace{14mu}{after}\mspace{14mu}{IV}} \\{{adminstration}\mspace{14mu}{of}\mspace{14mu}{parent}\mspace{14mu}\left( {{historic}\mspace{14mu}{data}} \right)}\end{matrix}}**{AUCtot}}\mspace{14mu}{ratio}} = \frac{{Total}\mspace{14mu}{AUC}\mspace{14mu}{of}\mspace{14mu}{parent}\mspace{14mu}{after}\mspace{11mu}{PO}\mspace{14mu}{administration}\mspace{14mu}{of}\mspace{14mu}{Prodrug}}{\mspace{14mu}\begin{matrix}{{Total}\mspace{14mu}{AUC}\mspace{14mu}{of}\mspace{14mu}{parent}\mspace{14mu}{after}\mspace{14mu}{PO}} \\{{adminstration}\mspace{14mu}{of}\mspace{14mu}{parent}\mspace{14mu}\left( {{historic}\mspace{14mu}{data}} \right)}\end{matrix}}}$

A dose escalation study D of one of the prodrugs (Ica) was carried outin rats in order to demonstrate the significant advantages of prodrugsover the parent for potential use in the treatment of HIV-1 patientsafter oral dosing. The exposure data and measured parameters from theprodrug dose escalation study were compared to similar data fromhistorical studies conducted with parent molecules.

FIGS. 3 and 4 compare the AUC (the area under the curve, a measure ofexposure to drug in the rat) of IVc from oral dosing of prodrug Ica(study D) to that obtained from dosing parent molecule IVc (Study E) anda rat toxicokinetic study (TK). Details for the historical IVc doseescalation (Study E) and the rat TK study (F) are shown in thesefigures.

As can be seen from FIGS. 3 and 4, the AUC and Cmax of parent moleculeafter oral administration of the prodrug (triangles) is greater thanthat which resulted from administration of the parent drug in twoseparate studies. Clearly, the data shows that in order to maximizeexposure multiples of drug in plasma, the prodrug offers a surprisingadvantage. Since the chemical structures of this class of molecules aresimilar, prodrugs of the class are expected to show enhancement inexposure from administration of prodrugs rather than parent. Given theuncertainty of improving oral exposure with phosphate prodrugs and thenovelty of the new compounds, this result was not obvious and issurprising in its magnitude.

TABLE 5 Rat TK Study; Study F; Historical Study of PO IVc in Rats atThree Doses Compound IVc (Historical Data) Dose (mg/kg) 15 75 200 (soln)(susp) (susp) Vehicle 80/10/10 PEG-400/Ethanol/0.1N NaOH Mean Cmax (μM)42 65  76 Mean 418  792  1077  AUC0-24 hr (μM * hr) Dose Ratio 1:5:13Cmax Ratio 1:1.5:1.8 AUC Ratio 1:1.9:2.6IV and Oral Rat PK Study Protocol of Phosphate Prodrugs Studies A, B,and C

Compounds Ic (phosphate prodrug of IVc), Ib (phosphate prodrug of IVb),and Ia (phosphate prodrug of IVa) were administered separately to groupsof three male Sprague-Dawley rats by IV bolus (1 mg/kg; all doses listedin this document were parent compound equivalent) or oral gavage (5mg/kg). The rats for oral dosing studies were fasted overnight. CompoundIc was administered as a free acid, whereas the other two were as sodiumsalts. The dosing solutions of all three prodrugs for both IV and oraladministration were prepared in 100% normal saline at 1 mg/mL (dosingsolution concentrations were parent compound equivalent). Plasma sampleswere collected in EDTA vacutainers over 24 hrs, and analyzed by LC/MS/MSfor both the prodrugs and parent molecules. Pharmacokinetic analysis wasperformed on Kinetica™.

Procedures for LC/MS/MS analysis are shown in the protocols below.

The results of this study are shown in Tables 2-4, middle two columns.

Oral Dose Escalation Study Protocol of Ica in Rats (Study D)

Groups of three fasted male Sprague-Dawley rats were orally administeredcompound Ica (disodium salt) at 4.5, 21, and 163 mg/kg (doses were IVcequivalent). The dosing solutions were prepared in water at 1, 5, and 20mg (compound Ic (free acid) equivalent)/mL for the doses of 4.5, 21, and163 mg (compound IVc equivalent)/kg, respectively. Plasma samples werecollected in EDTA vacutainers over 24 hrs, and analyzed by LC/MS/MS forboth Ic and IVc. Pharmacokinetic analysis was performed on Kinetica™.

The results of this study are shown in Table 46 and FIGS. 3-5.

TABLE 46 Oral Rat Dose Escalation Study Dosing of Ica IVc afterphosphate prodrug Ica (Na salt) IVc (n = 2) (all doses are IVcequivalent) (n = 3) Historical Data Dose (mg/kg) 5 25 200 25 75 200(soln) (soln) (soln) (soln) (susp) (susp) Vehicle Water 80/10/10PEG-400/Ethanol/ 0.1N NaOH Particle size Soln Soln Soln Soln 27 31 (μm)Mean Cmax (μM) 29 ± 14 98 ± 21 281 ± 55 46 86 42 C-24 hr (μM) 0.029 ±0.008 0.35 ± 0.13 58 ± 36 0.61 5.2 10 (n = 1) Mean AUCtot 109 ± 15  586± 53  2925 ± 304* 458 1071 518* (μM * hr) Mean Tmax (hr) 0.50 ± 0.25 1.7± 2.0  1.1 ± 0.80 4.0 5.0   4.0 Mean T    2.3 ± 0.07  2.5 ± 0.15  13 ±7.5 3.4 6.8 20 (hr) Dose Ratio 1:5:40 1:3:8 Avg. Cmax Ratio 1:3.4:9.71:1.9:0.91 Avg. AUC Ratio 1:5.4:27 1:2.6:1.1* 1. *The AUC was from 0-24hr since the AUC (24 hr-infinity) was at least greater than 35% of thetotal AUC (0-infinity). This portion was too great to have an accuratemeasure of the total AUC. 2. The AUC conversion ratio at 5 mg/kg of IVcfrom Ica, calculated as the ratio of IVc AUC after Ica dosing divided byIVc AUC after direct dosing of IVc, was 0.99. 3. The prodrug Ica wasonly detected at ~20-40 nM in one to two samples in each rat in the 200mg/kg dose group.In Vivo MethodsProcedure for Study A1: PO and IV Administration of IVa to Rats; PO Dosewas 5 mg/kg

Compound IVa was administered in a polyethylene glycol 400(PEG400)/ethanol (EtOH) solution (90/10, v/v) unless noted otherwise.Plasma and tissue samples were collected and stored at −20° C. untilanalysis. Male Sprague-Dawley rats (300-350 g, Hilltop Lab Animals,Inc., Scottdale, Pa. 15683) with cannulas implanted in the jugular veinand/or bile duct were used in the pharmacokinetic studies of compoundIVa. Rats were fasted overnight in PO studies. Blood samples (0.3 mL)were collected from the jugular vein in EDTA-containing microtainertubes (Becton Dickinson, Franklin Lakes, N.J. 07147) to obtain plasma.In the IV studies, a dose of 1 mg/kg was administered to 3 rats over 0.5min, and serial plasma samples were collected before dosing and 2, 10,15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing. In the POstudies, rats (n=3) received PO doses of 5 and 100 mg/kg. Serial plasmasamples were taken before dosing and 15, 30, 45, 60, 120, 240, 360, 480,and 1440 min after dosing.

The oral (PO) results of this study are shown in the last right mostcolumn of Table 2.

Parent Drug Study B1

For the IV and PO pharmacokinetic studies of COMPOUND IVb in rats,COMPOUND IVb was dissolved in PEG-400/ethanol (90/10) as a solution. Forthe IV and PO pharmacokinetic studies of COMPOUND IVB in dogs, COMPOUNDIVb was dissolved in PEG-400/ethanol (90/10) with pH adjustment with 0.1N NaOH. Details of the formulations are provided in Table 6.

Rat.

Male Sprague-Dawley rats (300-350 g, Hilltop Lab Animals, Inc.,Scottdale, Pa.) with cannulas implanted in the jugular vein and/or bileduct were used. The rats were fasted overnight in the PO pharmacokineticstudies. Blood samples of 0.3 ml were collected from the jugular vein inEDTA-containing microtainer tubes (Becton Dickinson, Franklin Lakes,N.J.), and centrifuged to separate plasma.

In the IV study, COMPOUND IVb was delivered at 1 mg/kg as a bolus over0.5 min (n=3). Serial blood samples were collected before dosing and 2,10, 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing.

In the PO study of COMPOUND IVb, the rats (n=3) received an oral dose of5 mg/kg of COMPOUND IVB. Serial blood samples were collected beforedosing and 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min afterdosing.

The results of this oral (PO) study are shown in Table 3, right mostcolumn.

TABLE 6 Formulations of IVb for in vivo Studies Compound Conc. IVbStudies Form Vehicle (mg/ml) Particle Size Rat PK amorphous 90:10,PEG400/ 3 NA (soln) (Study B1) EtOH adj. to pH 8.6-9.0Bioanalytical Methods for In Vivo Studies Analyzing for IVb

This refers to method for analyzing for concentration levels of IVb inrat plasma samples (used for studies B and B1).

Quantitation of Compound IVb by LC/MS/MS in Plasma.

Aliquots of plasma samples from rat, dog, monkey or chimpanzee studieswere prepared for analysis by precipitating plasma proteins with twovolumes of acetonitrile containing the internal standard, COMPOUND IVa.The resulting supernates were separated from the precipitated proteinsby centrifugation for 10 minutes and transferred to autosampler vials.Samples were either prepared manually, or with the use of the Tomtecautomated liquid handler. An aliquot of 5 μL was injected for analysis.

The HPLC system consisted of two Shimadzu LC10AD pumps (Columbia, Md.),a Shimadzu SIL-HTC autosampler (Columbia, Md.), and a Hewlett PackardSeries 1100 column compartment (Palo Alto, Calif.). The column was a YMCPro C18 (2.0×50 mm, 3 μm particles, Waters Co., Milford, Mass.),maintained at 60° C. and a flow rate of 0.3 ml/min. The mobile phaseconsisted of 10 mM ammonium formate and 0.1% formic acid in water (A)and 100% 10 mM ammonium formate and 0.1% formic acid in methanol (B).The initial mobile phase composition was 95% A. After sample injection,the mobile phase was changed to 15% A/85% B over 2 minutes and held atthat composition for an additional 1 minute. The mobile phase was thenreturned to initial conditions and the column re-equilibrated for 1minute. Total analysis time was 4 minutes.

The HPLC was interfaced to a Micromass Quattro LC. Ultra high puritynitrogen was used as the nebulizing and desolvation gas at flow rates of100 L/hr for nebulization and 1100 L/hr for desolvation. The desolvationtemperature was 300° C. and the source temperature was 150° C. Dataacquisition utilized selected reaction monitoring (SRM). Ionsrepresenting the (M+H)⁺ species for IVb and the internal standard wereselected in MS1 and collisionally dissociated with argon at a pressureof 2×10⁻³ torr to form specific product ions which were subsequentlymonitored by MS2. The transitions, voltages and retention times aresummarized in Table 7.

TABLE 7 Parameters for MS/MS Analysis of COMPOUND IVB and COMPOUND IVa(IS) COMPOUND IVB COMPOUND IVa SRM transition mz 448 > 105 423 > 205Cone Voltage (V) 22 30 Collision Energy (V) 16 30 Retention time(minutes) 2.6 2.4

The plasma standard curve ranged from 4 to 8000 ng/ml, the brain curvefrom 1-1000 ng/ml. The curves were fitted with a quadratic regressionweighted by reciprocal concentration (1/x). Standards were analyzed induplicate. Quality control (QC) samples, prepared in blank plasma, atthree concentrations within the range of the calibration curve were alsoanalyzed in triplicate with each plasma analytical set. For thiscompound, the predicted concentrations of 90% of the plasma QCs werewithin 20% of nominal concentration, indicating acceptable assayperformance.

Vehicles and Formulations.

Referring to Table 8, when the vehicle used contains NaOH, Compound IVcsolution formulations were pH adjusted using NaOH to obtain a pH of8.6-9.0, where the compound is partially ionized, based on it's pKa at8.4.

TABLE 8 Formulations of Compound IVc for InVivo Studies Compound Form-Conc. IVc Studies Lot State Vehicle (mg/ml) Particle Size Rat PK 02-001unknown 90:10, PEG400/EtOH 3 NA (Study C1) 02-003 adj. to pH 8.6-9.0Pre-Tox 02-004 crystalline 80:10:10 2.5 NA Escalating PEG400/EtOH/0.1N7.5 mean = 27.22 μm (95% Dose NaOH 20 <84.4 μm) (Study E) Multi-ModalDistribution, 3 peaks Overall mean = 31.07 μm (95% <157.7 μm) Pre-ECN02-005 unknown 80:10:10 3 NA Rat Tox PEG400/EtOH/0.1N 15 Multi-ModalDistribution, 2 (Rat TK) NaOH 40 peaks (Study F) Overall mean = 19.25 μm(95% <52.3 μm) Multi-Modal Distribution, 2 peaks Overall mean = 21.78 μm(95% <57.5 μm)Quantitation of Compound IVc by LC/MS/MS in Plasma (Used in Studies C,C1 and D).

Aliquots of plasma samples from rat, dog, monkey and chimpanzee studieswere prepared for analysis by precipitating plasma proteins with twovolumes of acetonitrile containing the internal standard, compound IVa.The resulting supernates were separated from the precipitated proteinsby centrifugation for 10 minutes and transferred to autosampler vials.Samples were either prepared manually, or with the use of the Tomtecautomated liquid handler. The HPLC system consisted of two ShimadzuLC10AD pumps (Columbia, Md.), a Shimadzu SIL-HTC autosampler Columbia,Md.), and a Hewlett Packard Series 1100 column compartment (Palo Alto,Calif.). The column was a YMC Pro C18 (2.0×50 mm, 3 μm particles, WatersCo., Milford, Mass.), maintained at 60° C. and a flow rate of 0.3ml/min. The mobile phase consisted of 10 mM ammonium formate and 0.1%formic acid in water (A) and 100% 10 mM ammonium formate and 0.1% formicacid in methanol (B). The initial mobile phase composition was 95% A.After sample injection, the mobile phase was changed to 15% A/85% B over2 minutes and held at that composition for an additional 1 minute. Themobile phase was then returned to initial conditions and the columnre-equilibrated for 1 minute. Total analysis time was 4 minutes.

The HPLC was interfaced to a Micromass Quattro LC. Ultra high puritynitrogen was used as the nebulizing and desolvation gas at flow rates of100 L/h for nebulization and 1100 L/h for desolvation. The desolvationtemperature was 300° C. and the source temperature was 150° C. Dataacquisition utilized selected reaction monitoring (SRM). Ionsrepresenting the (M+H)⁺ species for IVc and the internal standard wereselected in MS1 and collisionally dissociated with argon at a pressureof 2×10⁻³ torr to form specific product ions which were subsequentlymonitored by MS2. The transitions, voltages and retention times aresummarized in Table 9.

TABLE 9 Parameters for MS/MS Analysis of IVc and IVa (IS) IVc IVa SRMtransition mz 474 > 256 m/z 423 > 205 Cone Voltage (V) 22 30 CollisionEnergy (V) 22 30 Retention time (minutes) 2.5 2.4

The plasma standard curve ranged from 4 to 8000 ng/ml, the brain curvefrom 1-1000 ng/ml. The curves were fitted with a quadratic regressionweighted by reciprocal concentration (1/x). Standards were analyzed induplicate. Quality control (QC) samples, prepared in blank plasma, atthree concentrations within the range of the calibration curve were alsoanalyzed in triplicate with each plasma analytical set. For thiscompound, the predicted concentrations of 90% of the plasma QCs werewithin 20% of nominal concentration, indicating acceptable assayperformance.

Quantitation of Three Pro-Drugs and their Parent Compounds by LC/MS/MSin Biological Matrices

This LC/MS/MS assay was developed to investigate three pro-drugcompounds and their respective parent molecules in biological matrices.The three pro-drug compounds were: Compounds Ia, Ic, and Ib and theirother respectives salts or free acids. Note this assay is used for thefree acid and salt forms of the three prodrugs as molecular ion detectedis independent of salt counterion. Their respective parent compoundswere: IVa, IVc, and IVb.

The HPLC system consisted of Shimadzu LC10ADvp pumps (Columbia, Md.) andHTC PAL autosampler (Leap Technologies, Cary, N.C.) linked to a SynergiHydro-RP analytical column (2.0×50 mm, Phenomenex, Torrance, Calif.).Mobile phase A consisted of 0.1% Formic Acid in water; mobile phase Bwas 0.1% formic acid in acetonitrile. LC flow rate was 0.4 mL/min intothe mass spectrometer. The initial mobile phase composition was 10% Bramped to 75% B over 1.75 min, held at that composition for 0.25 min,ramped to 100% B over 0.1 min, held for 0.6 min, returned to initialconditions over the next 0.1 minute and then re-equilibrated. Totalanalysis time was 4 min. Retention times for all analytes ranged between1.5 and 2.6 min.

The HPLC system was interfaced to a Sciex API3000 triple quadrupole massspectrometer (Toronto, Canada) equipped with the Turboionspray sourceset at 450° C. and ionspray voltage set to 4.5 kV. UHP nitrogen was usedas nebulizer and auxiliary gases with pressures of 80 psi and 7 L/min,respectively. The analysis was performed in positive ion mode. Thetransitions monitored for all compounds and their collision energies(CE) were: m/z 533.41>435.24 for Ia (CE=19); m/z 584.46>486.29 for Ic(CE=23); m/z 558.31>432.13 for Ib (CE=19); 423.39>204.96 for IVa(CE=31); 474.36>255.97 for IVc (CE=29); 448.35>105.20 for IVb (CE=35).

To accommodate a wide variety of biological sample matrices,acetonitrile precipitation was used in sample preparation. Test samplesand standards were transferred to a 96 well plate using the PackardMultiprobe II (Packard Instruments, Downers Grove, Ill.). 200 μL ofacetonitrile containing the internal standard (BMS-647257, 500 nM) wasadded to 100 μL aliquots of both test samples and standards in the 96well plate using the Tomtec Quadra 96. The plate was then vortexed forapproximately 3 minutes and centrifuged at 3000 rpm for 15 minutes.Using the Tomtec Quadra 96, 150 μL of supernatant was transferred fromthe plate to a clean 96 deep well plate. 150 μL of 0.2% formic acid inwater was then added to each well using the Tomtec Quadra 96 and theplate was vortexed before analysis.

Standard curves at eight concentration points from 5 nM to 10 μM wereprepared from stock solutions in acetonitrile and serially diluted inmatrix for both pro-drug and parent compounds. Standard curves weretransferred in duplicate 100 uL aliquots to a 96 well plate containingthe test samples, extracted with the test samples as described above,and injected at the beginning, middle, and end of the analyticalsequence. The standard curves were fitted with a linear regressionweighted 1/x². Data and chromatographic peaks were processed andconcentrations of standards and unknowns were quantitated usingPEBiosystems Analyst™ 1.1.

In Vivo Methods

Conditions for Study C1, E and F (Rat PK, MAP and TK Studies)

For the IV and PO pharmacokinetic studies of compound IVc in rats,compound IVc was dissolved in PEG-400/ethanol (90/10) as a solution.Please refer to Table 8.

Rat.

Male Sprague-Dawley rats (300-350 g, Hilltop Lab Animals, Inc.,Scottdale, Pa.) with cannulas implanted in the jugular vein and/or bileduct were used. The rats were fasted overnight in the PO pharmacokineticstudies. Blood samples of 0.3 ml were collected from the jugular vein inEDTA-containing microtainer tubes (Becton Dickinson, Franklin Lakes,N.J.), and centrifuged to separate plasma.

In an IV study, compound IVc was delivered at 1 mg/kg as a bolus over0.5 min (n=3). Serial blood samples were collected before dosing and 2,10, 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing.

PO Single Dose Study C1

In the PO study Cl of Compound IVc, the rats (n=3) received an oral doseof 5 mg/kg of Compound IVc. Serial blood samples were collected beforedosing and 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min afterdosing. The oral (PO) results from Study Cl are in Table 4, right mostcolumn.

Oral (PO) Dose Escalation Study E

In the oral dose escalation study E of Compound IVc, groups of rats (n=2per group) received oral doses of 25, 75, and 200 mg/kg. At 25 mg/kg,the dosing solution was in solution; at the higher doses, the dosingsolutions were suspensions. Serial blood samples were collected beforedosing and 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min afterdosing. Brain samples were collected at 1440 min to assess brainpenetration. The brain samples were blotted dry and the wet weights wererecorded. The oral (PO) results from Study E are in Table 46.

2-Week Oral Dose Rat Toxicology Study F

In the 2-week oral rat study F (n=six/sex/group), Compound IVe wasadministered at daily doses of 15, 75, or 200 mg/kg. Toxicokineticevaluations on days 1 and 14 indicated that systemic exposures(AUC_(0-24h)) of Compound IVe were generally dose related but were notdose proportional (see Table 5), with no evidence of autoinduction oraccumulation. On Day 14, AUC_(0-24h) values were slightly higher infemales (≦644 μg*hr/ml) compared to males (≦526.88 μg*hr/ml) atdosages≦200 mg/kg/day. The oral (PO) results from Study F are in Table5.

Map Study G PO and IV Dosing Study of Diester IIa in Rats Structure ofCompound IIa

The procedure for the oral dosing leg of MAP study A1 was followedexcept that diester IIa was utilized in place of Compound IVa. Analysisfor compound IVa showed that dosing of the diester IIa by oral routeproduced substantial IVa. The data is described in Table 10 below:

TABLE 10 for Map Study G Dose and Route: Data measured for IVa followingPO dosing of PO 5 mg/kg diester IIa (di-tert butyl ester prodrug of IVa)Cmax p.o. (nM) 1123 ± 270 (′IVa conc) Tmax p.o. (hr)   2.7 ± 1.2 (IVa) F(%) 33 (IVa) AUC p.o. (μM * hr)   4.0 ± 1.0 (IVa) Cp @ 24 hr p.o. (nM)Not detected T½ p.o. (hr)   1.3 ± 0.26 (IVa)

Map Study H PO and IV Dosing Study of Monoester II′a in Rats Structureof Compound II′a

The procedure for the oral dosing leg of MAP study A1 was followedexcept that monoester II′a was utilized in place of Compound IVa.Analysis for compound IVa showed that dosing of the monoester Ira byoral route produced substantial IVa. The data is described in the tablebelow:

TABLE 11 for Map Study H Data measured for IVa following PO dosing ofDose and Route: monester II′a (mono-tert butyl ester prodrug of PO 5mg/kg IVa) Cmax p.o. (nM) 1586 ± 615 (IVa conc) Tmax p.o. (hr)   2.0 ±1.7 (IVa) F (%) 44 (of IVa) AUC p.o. (μM * hr)   5.9 ± 2.2 (IVa) Cp @ 24hr p.o. (nM)  3.61 (n = 2/3) T½ p.o. (hr)   2.8 ± 1.6 (IVa)

A significant number of studies were done to demonstrate andcharacterize the surprising utility of the prodrugs I. Dose escalationexposure experiments comparing exposure of parent molecules after dosingprodrug and parent were carried out in rats and dogs for prodrugs I ofparent molecules IVa, IVb, and IVc. The effect of food and dose onexposure of IVa after dosing prodrug Iab or parent IVa was compared indogs. The prodrug showed suprising ability to improve exposure and avoideffects of feed as compared to parent. Low dosage full pharmacokineticstudies (oral and IV dosing) were carried out in rats dogs and monkeysfor prodrugs Iab, Ibb, and Icb to show conversion to parent compoundsIVa, IVb, and IVc respectively. Oral dosing studies in rats were carriedout for sprodrug Ie (free acid), and for parent compound IVf todemonstrate conversion and systemic exposure of parents IVe and IVfrespectively. Data is shown above or below in this application.

Additional Profiling Section 1

Additional Studies with Iab:

Iac is the free acid phosphate prodrug of the N-hydroxymethyl adduct ofIVa and is hydrolyzed by alkaline phosphatase (ALP) to form IVa. Iab, amono-lysine salt of Iac, was used for all of the following studies.

IV and PO pharmacokinetic studies of Iab were conducted in rats, dogsand monkeys. In all cases, blood samples were collected in the presenceof EDTA. The presence of EDTA, a known ALP inhibitor, minimizedsignificant ex vivo conversion of Iab during sample processing. IVa wasrapidly formed following IV administration of Iab. Good oralbioavailabilities (62-94%) of IVa were observed after administration ofIab in rats, dogs and monkeys with very little or no Iab present inplasma. Since there are high levels of ALP expression in the gut, it islikely that following oral administration of Iab, Iab is hydrolyzed byALP present at the brush border membranes of the intestinal lumen toform IVa, which is rapidly absorbed due to its high permeability.

In vitro incubation studies were conducted for a qualitative assessmentof ALP-dependent hydrolysis of Iab in different tissues. Iab washydrolyzed in the presence of serum and hepatocytes from rat, dog,monkey and human, as well as human placental ALP. On the occasions whereIab and IVa were measured, the conversion of Iab to IVa was nearstoichiometric. Due to hydrolysis in serum, the protein binding of Iabcould not be determined. Based on the in vitro data, it is anticipatedthat Iab will be hydrolyzed by human ALP and that IVa will be formedafter oral administration of Iab to human subjects.

The crystalline solubility of Iab at room temperature increases from0.22 mg/ml at pH 1.4 to >12 mg/ml at pH 5.4 and pH 8.9; aqueoussolutions containing >100 mg/mL have been prepared for in vivotoxicology studies. In comparison, the aqueous solubility of the parentcompound, IVa, at room temperature as crystalline material wasdetermined to be 0.04-0.9 mg/mL (pH range of 1.5-10). Iab exhibitsacceptable solution and solid stabilities.

The higher aqueous solubility of Iab provides a means to overcome thedissolution-rate limited absorption of IVa below certain doses andthereby increased the exposures of IVa in oral dose escalation andtoxicokinetic studies. Iab, when dosed orally at ˜200 mg/kg of IVaequivalent, provided 2-fold higher AUC of IVa in rats and dogs, withoutsignificant plasma exposure to the prodrug, as compared to the AUC fromthe historical IVa suspension studies at the similar dose. Moreover, theAUC and Cmax values of IVa in fasted dogs receiving Iab dry-filledcapsules (200 mg/dog of IVa equivalent) were 38 and 58 times,respectively, those attained in fasted dogs given the IVa clinicalcapsule formulation, and 4 and 6 times, respectively, those attained infed dogs given the IVa clinical capsule formulation.

No significant differences in AUC and Cmax of IVa were observed betweenfasted and fed dogs receiving Iab, whereas a 9-fold improvement wasobserved in fed dogs as compared to fasted dogs receiving IVa. Thesedata suggest that efficacious blood levels of IVa may be achieved inHIV-infected patients without the requirement for a high fat meal. Thespray dried form of IVa gave rise to similar exposure levels of IVa asthat observed from Iab in dogs.

Single-Dose Toxicokinetic Tolerability Study in CD Rats

A 1-day oral toxicokinetic study in rats was conducted using the prodrugIab (monolysine salt). Iab was administered at dosages of 16, 72, and267 mg/kg (free acid) by oral gavage to three male rats/group usingwater as the vehicle (solution formulation). The dosages of prodrug freeacid correspond to IVa (parent) molar equivalent dosages of 13, 57, and211 mg/kg, respectively. The endpoint evaluated was plasmatoxicokinetics of Iab and IVa in the individual rats.

The mean toxicokinetic values are provided in Table 12.

TABLE 12 Mean toxicokinetic values for Iab and IVa in male rats given≦267 mg/kg of Iab as a single oral dose Dosages (mg/kg) Iab 16 72 267IVa 13 57 211 molar equiv. Iab IVa Iab IVa Iab IVa Cmax (μM) 0.068 260.095 104 0.11 214    C_(24 h) (μM) <LLQ¹    0.090 <LLQ     0.027 <LLQ1.9 AUC (μM · h) 0.020²  67³ 0.049²  261³ 0.057² 1161³     T   (h) 0.64  3.2 0.37    2.3 0.045 3.4 Values represent means from 1-3 rats/groupfor Iab data and 3 rats/group for IVa data. ¹Below the lower limit ofquantification. ²AUC from zero to time of last quantifiable sample ³AUCfrom zero to ∞

Mean maximum plasma concentration (Cmax) of both Iab (prodrug) and IVa(parent) was achieved within 1.1 hour post-dose. The plasma area underthe plasma concentration-time curve (AUC) of the prodrug was ≦13.03%that of the parent. (In rats given ≦267 mg/kg of Iab, the AUC of IVaincreased proportionally with Iab dosage, and Cmax increased in a lessthan dosage-proportional manner between 72 and 267 mg/kg of Iab.

A comparison of IVa AUC obtained in rats given either IVa (2) or Iab, isshown in FIG. 6.

Solubility in preclinical formulations has been an issue. The AUC of IVais equivalent for both parent and prodrug at lower dosages (e.g., ≦50mg/kg) because both were formulated as solutions (PEG-400 for parent andwater for prodrug) but at a high dosage (e.g., 200 mg/kg) the neutralparent was formulated as a suspension whereas the prodrug salt wasformulated as an aqueous solution.

A 2-week rat toxicity study using dosages of 5, 50, or 500 mg/kg BID tosupport the IND is ongoing (3). The in-life phase of the study has beencompleted, and there were no noteworthy in-life observations.

Single-Dose Toxicokinetic and Tolerability Study in Dogs

A multi-phase study was conducted to evaluate the tolerability of theprodrug, Iab (monolysine salt) at dosages of 24, 90, or 240 mg/kg (freeacid, molar equivalent to 19, 71, or 190 mg/kg of parent IVa,respectively) and the toxicokinetics of Iab and IVa (4). Iab wasadministered to two female dogs/group once daily either as an aqueoussolution (24 or 90 mg/kg) or dry-filled capsules (24, 90 [once and twicedaily] or 240 mg/kg). The endpoints were: clinical signs, body weight,food consumption, and plasma toxicokinetics of Iab and IVa. In allcases, a 1-week washout period was used between doses for all phases ofthe study.

The plasma toxicokinetic values from the initial phase of the study areshown in Table 13.

TABLE 13 Toxicokinetic values for IVa in female dogs given ≦90 mg/kg ofIab as a single oral dose Dosages (mg/kg) Ibb 24 90 24 IVa molar 19 7119 equiv. Formulation Solution Solution Dry-filled capsule Dog Dog DogDog Dog Dog #1201 #1202 #3201 #3202 #4201 #4202 Cmax (μM) 72.3 66.5 124125 79.7 85.6 C_(24 h) (μM) 0.61 0.33 3.2 2.2 1.4 0.87 AUC_(0-∞) 465 330844 838 486 514 (μM · h) T   (h) 3.2 3.1 4.4 3.9 4.2 3.4

Iab was not detected in the plasma samples. Mean maximum plasmaconcentration (Cmax) of IVa was achieved between 1-2 hours post-dose.When Iab was given as a solution, both the Cmax and AUC increased inless than dosage proportional manner between 24 and 90 mg/kg. Emesis wasobserved at about 30 minutes after dosing in both dogs given 90 mg/kg.The Cmax and AUC of IVa were equivalent following Iab administration at24 mg/kg using either dry-filled capsules or an aqueous solution.

Other than emesis, there were no clinical signs observed, and there noeffects on body weight and food consumption.

To determine whether emesis could be eliminated/reduced byadministration of Iab as a dry-filled capsules, the next phase of thestudy was conducted using dosages of 90 or 240 mg/kg, and 90 mg/kg giventwice 4 h apart (BID). The toxicokinetic values are shown in Table 14.

TABLE 14 Toxicokinetic values for IVa in female dogs given ≦240 mg/kg ofIab as a single oral dose or with twice daily dosing (BID) Dosages(mg/kg) Ibb 90 240 180 (90 BID) IVa molar 71 190 142 (71 BID) equiv.Formulation Solution Solution Dry-filled capsule Dog Dog Dog Dog Dog Dog#1201 #1202 #2201 #2202 #3201 #3202 Cmax (μM) 214 152 172 189 311 248C_(24 h) (μM) 4.6 0.89 2.8 6.6 34 50 AUC_(0-∞) 1740 960 1186 1584 3305¹2485 (μM · h) T   (h) 3.6 2.9 3.7 4.5 6.1 13 ¹AUC from zero to 24 h

There was no difference in Cmax or AUC between dogs given 90 or 240mg/kg of Iab administered by dry-filled capsules. Emesis was observed inDogs #1201, #2201, and #2202 about 1 hour after dosing. The vomit wascollected and assayed for tab content to estimate the amount of thetotal dose that was lost; the percentage of estimated total dose lostwas <1% for #1201, ≈90% for #2201, ≈9% for #2202. Although theestimations of “dose lost” do not appear to be quantitatively consistentwith the plasma AUC data, it does indicate that test article can befound in vomit within a short time after dosing. Iab was detected inplasma of the dogs, 0.005-0.049 μM at 1 hour post dose and 0.005-0.006μM at 2 hours post dose; the prodrug was not detectable at later timepoints.

A comparison of IVa AUC obtained in dogs given either IVa (5, 6) or Iab,is shown in FIG. 7.

Vehicles and Formulations

Summary of Formulations Used for Key PK and Safety Studies

All in vivo PK studies in rats, dogs, and monkeys were performed usingaqueous solutions for PO and IV dosing. Toxicology and exposure studiesin dogs were performed with aqueous solutions at dosages of 24 and 90mg/kg and drug in capsule formulations at dosages of 24, 90, and 240mg/kg of Iab, mono-lysine salt.

In the rat oral dose escalation study, Iab was dosed as aqueoussolutions at concentrations of 4.5, 20.0, and 73.5 mg/mL (mono-lysinesalt form of prodrug). A significant improvement in AUC and Cmax of IVa,parent, after oral dosing of Iab, prodrug, were observed compared to thehistorical data of IVa oral dosing.

Drug in capsule formulations of Iab at doses of 20 mg/kg of IVaequivalent were used for food effect studies in dogs. The prodrug wascompared to the clinical capsule formulation of the parent compound,IVa, at 20 mg/kg. When IVa clinical capsule was dosed, a 9-fold increasein exposure was seen in dogs fed a high fat meal compared to fasteddogs. Upon dosing of the prodrug, Iab, the exposure of IVa wassignificantly higher and as expected, the exposure was not significantlydifferent between fasted and fed dogs.

Metabolism and Pharmacokinetics Summary

Summary of Findings and Interpretation

Iab is the phosphate prodrug of the N-hydroxymethyl adduct of IVa and ishydrolyzed by alkaline phosphatase (ALP) to form IVa. Afteradministration of Iab to animals, therefore, plasma samples wereprepared from blood collected in the presence of EDTA, a known ALPinhibitor. Conversion of Iab to IVa was minimal (<2%) in rat, monkey andhuman blood containing EDTA, and approximately 6% in dog bloodcontaining EDTA. No significant ex vivo conversion of Iab is expectedduring sample storage (−20° C.) and analysis of Iab.

The hydrolysis of Iab was studied in animal and human in vitro systems.Since multiple ALP isoforms are widely distributed in various tissues,quantitative in vitro to in vivo correlations were not attempted(Fishman et al., 1968; Komoda et al., 1981; Moss, 1983; Yora andSakagishi, 1986; Sumikawa et al., 1990). Therefore, the studies werelimited to a qualitative assessment of ALP-dependent hydrolysis indifferent tissues. Iab was hydrolyzed in the presence of serum andhepatocytes from rat, dog, monkey and human, as well as in humanplacental ALP. On the occasions where Iab and IVa were measured, theconversion of Iab to IVa was near stoichiometric. Due to hydrolysis inserum, the protein binding of Iab could not be determined.

None or very low levels of Iab were detected in rat, dog and monkeyplasma after oral administration of Iab. IVa was rapidly formedfollowing IV administration of Iab in rats, dogs and monkeys. The IV AUCconversion ratios were 1.5 in rats, 0.80 in dogs and 0.70 in monkeys,suggesting good conversion from Iab to IVa.

Good oral bioavailabilities (62-94%) of IVa were observed afteradministration of Iab in rats, dogs and monkeys. More significantly, thehigher aqueous solubility of Iab lessened the dissolution-rate limitedabsorption of IVa below certain doses and thereby increased theexposures of IVa in oral dose escalation and toxicokinetic studies(Tables 12-14 and FIG. 6-7). The AUC levels of IVa in fasted dogsreceiving Iab capsules were about 40 times the levels in fasted dogsgiven IVa clinical form capsules. Although a 40-fold difference islikely an over-prediction of the clinical situation, based on thedevelopment experience with IVa, Iab clearly demonstrated the potentialto improve the dissolution-rate limited absorption seen with parent IVa.

To investigate the effect of food on the oral absorption of IVa in dogs,Iab and IVa were administered in capsules under fasting and fedconditions. No significant differences in AUC and Cmax were observedwith Iab, whereas a 9-fold improvement was observed with IVa uponfeeding. These data suggest potential clinical benefits for HIV-infectedpatients, in whom efficacious blood levels of IVa may be achievedwithout the requirement for a high fat meal.

Methods

The studies described in this report used the mono-lysine salt of Iab,unless stated otherwise.

Quantitation of Iab and IVa by LC/MS/MS

An LC/MS/MS method was developed for the analysis of Iab and IVa inplasma samples from the animal pharmacokinetic studies as well as inacetonitrile supernatant from in vitro incubation studies. For theanalysis in plasma, a Packard Multiprobe instrument was used to transfer50 μL of each standard, QC, and plasma sample to a clean 96-well platefor protein precipitation extraction. After the addition of 200 μL ofacetonitrile containing the internal standard IVc, the samples werevortex mixed and the resulting supernatant was separated from theprecipitated proteins by centrifugation for 10 min. For the analysis inthe supernatant generated from the in vitro studies, an equal volume ofthe supernatant and acetonitrile containing the internal standard wasmixed. An aliquot of the above supernatant was transferred using aTomtec automated liquid handler to a second clean 96-well plate. Anequal volume of water was added, and the plate was capped and vortexmixed.

The HPLC system consisted of Shimadzu LC10ADvp pumps (Columbia, Md.) anda HTC PAL autosampler (Leap Technologies, Cary, N.C.) linked to aPhenomenex Synergi Fusion-RP analytical column (2.0×50 mm, 5μ; Torrance,Calif.). Mobile phase A consisted of 5 mM ammonium formate in water;mobile phase B was 100% acetonitrile. LC flow rate was 0.38 mL/min. Theinitial mobile phase composition was 3% B, ramped to 60% B over 1.75 minand held for 0.25 min, ramped to 100% B over 0.1 min and held for 0.8min, returned to initial conditions over the next 0.1 min, andre-equilibrated. Total analysis time was 4.0 min. The retention time forIab, IVa and IVc was 1.50, 1.67 and 1.73 min, respectively.

The HPLC system was interfaced to a Sciex API4000 triple quadrupole massspectrometer (Toronto, Canada) equipped with the Turboionspray sourceset at 550° C. and the ionspray voltage set to 4.5 kV. UHP nitrogen wasused as nebulizer and auxiliary gas with the pressure of 80 psi and 7L/min, respectively. The collision energies for Iab, IVa and IVc were21, 29 and 31 volts, respectively. Data acquisition utilized selectedreaction monitoring (SRM). Ions representing the positive ion mode(M+H)⁺ species for Iab, IVa and the internal standard were selected inMS1 and collisionally dissociated with nitrogen and optimized collisionenergies to form specific product ions subsequently monitored by MS2.The SRM transitions for Iab, IVa and IVc were m/z 533→435, 423→205 and474→256, respectively.

Standard curves ranging from 5 nM to 10 μM were prepared from stocksolutions and serially diluted in matrix for both Iab and IVa. Standardcurves were aliquoted in duplicate, extracted with the samples, andinjected at the beginning, middle, and end of the analytical sequence.The standard curves were fitted with a linear regression weighted byreciprocal concentration 1/x². Data and chromatographic peaks wereprocessed and concentrations of standards and unknowns were quantitatedusing PEBiosystems Analyst™ 1.1.

In Vitro Methods

(1) Stability of Iab in EDTA Blood, Serum and Tris-HCl Buffer

The stability of Iab was studied in fresh blood and serum from rat, dog,monkey and human (n=2). The blood was collected in vacutainerscontaining K₂EDTA (Becton Dickinson, Franklin Lakes, N.J.). The serumwas collected in vacutainers containing no anticoagulant. Iab wasincubated at a starting concentration of approximately 10 μM for 60-90min at 37° C. Serial samples were taken at the pre-determined times.Aliquots of blood samples (200 μL) were first mixed with 100 μL of waterfollowed by 400 μL of acetonitrile. The serum samples (50 μL) were addedinto microtainers containing K₂EDTA (Becton Dickinson, Franklin Lakes,N.J.) followed by the addition of 100 μL of acetonitrile. Thesupernatant was analyzed for both Iab and IVa by LC/MS/MS.

The stability of Iab was also evaluated, as described above, in Tris-HClbuffer (0.1 M, pH 7.5).

(2) Hydrolysis of Iab in the Presence of Human Placental ALP

Solid human placental ALP was obtained from Sigma (P-3895, St. Louis,Mo.). A solution of 1000 units/L was prepared in Tris-HCl buffer (0.1 M,pH 7.5). Solutions of 100 and 10 units/L were obtained by serialdilution. Iab was incubated in the 10, 100 and 1000 units/L solutions(n=2) at 37° C. for 2 hr. The starting concentration of Iab in theincubation was 10 μM. Aliquots of 100 μL samples were taken atpre-determined times and added into K₂EDTA microtainers followed by theaddition of 200 μL of acetonitrile. The supernatant was analyzed forboth Iab and IVa by LC/MS/MS.

In Vivo Studies

All blood samples (0.3 mL) were collected in microtainers containingK₂EDTA (Becton Dickinson, Franklin Lakes, N.J.) and placed on chippedice. After centrifugation, plasma was separated and stored at −20° C.until analysis. The mono-lysine salt of Iab (Form 3, Lot 1) was used forthe pharmacokinetic studies. The dosing solutions of Iab were preparedin either sterile water (for IV administration in dogs and monkeys) ordistilled water (for all other dose administration).

(1) In Vivo Studies in the Rat

Male Sprague-Dawley rats (300-350 g, Hilltop Lab Animals, Inc.,Scottsdale, Pa.) with cannulas implanted in the jugular vein were used.The rats were fasted overnight in the PO pharmacokinetic studies. Bloodsamples were collected from the jugular vein.

In the IV study, Iab was delivered at 1.4 mg/kg (free acid, or 1.1 mg/kgof IVa equivalent) as a bolus over 0.5 min (n=3). The concentration ofthe dosing solution was 1.4 mg/mL, and the dosing volume was 1 mL/kg.Serial blood samples were collected before dosing and at 2, 10, 15, 30,45 min, 1, 2, 4, 6, 8 and 24 hr after dosing.

In the PO study, Iab was administered at 7.9 mg/kg (free acid, or 6.3mg/kg of IVa equivalent) by oral gavage (n=3). The concentration of thedosing solution was 4.0 mg/mL, and the dosing volume was 2 mL/kg. Serialblood samples were collected before dosing and at 15, 30, 45 min, 1, 2,4, 6, 8 and 24 hr after dosing.

(2) In Vivo Studies in the Dog

The IV and PO studies of Iab were conducted in a crossover fashion inthree male beagle dogs (11±1.1 kg, Marshall Farms USA Inc., North Rose,N.Y.). There was a two-week washout period between the IV and POstudies.

In the IV study, Iab was infused via the cephalic vein at 1.2 mg/kg(free acid, or 0.95 mg/kg of IVa equivalent) over 5 min at a constantrate of 0.1 mL/kg/min. The concentration of the dosing solution was 2.4mg/mL, and the dosing volume was 0.5 mL/kg. Serial blood samples werecollected from the femoral artery before dosing and at 5, 10, 15, 30, 45min, 1, 2, 4, 6, 8 and 24 hr after dosing.

In the PO study, the dogs were fasted overnight before dosing. Iab wasadministered by oral gavage at 6.6 mg/kg (free acid, or 5.2 mg/kg of IVaequivalent). The concentration of the dosing solution was 13.2 mg/mL,and the dosing volume was 0.5 mL/kg. Serial blood samples were collectedbefore dosing and at 15, 30, 45 min, 1, 2, 4, 6, 8 and 24 hr afterdosing.

To study the effect of food on the oral absorption of IVa afteradministration of Iab and IVa, these two compounds were administered incapsules as solid to a group of three dogs in a cross-over fashion underovernight fasting and fed conditions. There was a one-week washoutperiod between each study. Iab was administered at 200 mg per dog (ca 20mg/kg) of IVa equivalent; IVa was administered in a clinical capsuleformulation at 200 mg per dog (ca 20 mg/kg). In the studies where thedogs were fed, the following meal was prepared: 2 slices of bacon, 2eggs, 2 pieces of toast with butter and jelly, 4 oz. hash browns and 8oz. of whole milk. After homogenization using a laboratory blender, themeal was equally divided into five portions and kept frozen. Before thestudy, the meals were thawed and each dog was fed one portion.

Additional formulation studies of IVa were conducted in fasted dogs (n=2per dose group). The dogs were administered either the clinical form orthe spray dried form of IVa in capsules. The clinical capsule form ofIVa was administered at a single dose of 20 mg/kg. The spray dried formof IVa was administered at 20, 75 and 200 mg/kg.

(3) In Vivo Studies in the Monkey

The IV and PO studies of Iab were conducted in a crossover fashion inthree male cynomolgus monkeys (11±1.2 kg, Charles River BiomedicalResearch Foundation, Houston, Tex.). There was a two-week washout periodbetween the IV and PO studies.

In the IV study, Iab was infused via the femoral vein at 1.3 mg/kg (freeacid, or 1.1 mg/kg of IVa equivalent) over 5 min at a constant rate of0.1 mL/kg/min. The concentration of the dosing solution was 2.6 mg/mL,and the dosing volume was 0.5 mL/kg. Serial blood samples were collectedfrom the femoral artery before dosing and at 5, 10, 15, 30, 45 min, 1,2, 4, 6, 8 and 24 hr after dosing.

In the PO study, the monkeys were fasted overnight before dosing. Iabwas administered by oral gavage at 7.1 mg/kg (free acid, or 5.6 mg/kg ofIVa equivalent). The concentration of the dosing solution was 14.2mg/mL, and the dosing volume was 0.5 mL/kg. Serial blood samples werecollected before dosing and at 15, 30, 45 min, 1, 2, 4, 6, 8 and 24 hrafter dosing.

(4) Data Analysis

All results are expressed as mean±SD, unless specified otherwise.

The pharmacokinetic parameters of Iab and IVa were calculated byNon-Compartmental Analysis using the KINETICAT™ software program(version 4.0.2, InnaPhase Co., Philadelphia, Pa.). The Cmax and Tmaxvalues were recorded directly from experimental observations. The AUC0-nand AUCtot values were calculated using the mixed log-linear trapezoidalsummations. The total body clearance (Cl), mean residence time (MRT),and the steady state volume of distribution (Vss) were also calculatedafter intravenous administration. The absolute oral bioavailability(expressed as %) was estimated by taking the ratio of dose-normalizedAUC values after oral doses to those after intravenous doses.

The hepatic clearance Cl_(H) was calculated from the following equationusing the well-stirred model:

${{Cl}_{H}\left( {{mL}\text{/}\min\text{/}{kg}} \right)} = \frac{{Qh} \times {Cl}_{{{int} \cdot {in}}\mspace{14mu}{vivo}}}{{Qh} + {Cl}_{{{int} \cdot {in}}\mspace{14mu}{vivo}}}$where Qh is the liver blood flow of 55, 31, 44 and 21 mL/min/kg for therat, dog, monkey and human, respectively (Davis and Morris, 1993).

The hepatic extraction ration (ER) was calculated at follows:ER=Cl_(H) /QhStudent's t-test was used for statistical analysis (Microsoft® Excel,Redmond, Wash.). Differences were considered statistically significantat the level of P<0.05.In Vitro StudiesStability of Iab in EDTA Blood, Serum and Tris-HCl Buffer

As part of the analytical assay validation, the stability of Iab wasstudied in blood containing EDTA, which is known to be an inhibitor ofalkaline phosphatases (Bowers, Jr. and McComb, 1966; Yora and Sakagishi,1986). After incubation at 37° C. for 60 min, there was 1.2% conversionfrom the initial concentration of Iab to IVa in the rat blood (Table15), and less than 1% conversion in the monkey and human blood (Tables15 and 16). There was approximately 6% conversion in the dog blood, andthe percentages of conversion were similar between two different dogs,as well as in the same dog on two different test occasions (Tables 15and 16). Under the sample storage condition of −20° C., the above smallpercentages of conversion observed at 37° C. are not expected tointroduce any significant ex vivo conversion during the analysis of Iab.

Iab was stable in the Tris-HCl buffer at 37° C. during the 60-min studyperiod (Table 17).

TABLE 15 Stability of Iab in the Fresh EDTA Blood from Rat, Dog andMonkey Rat Blood (n = 2) Dog A Blood (n = 2) Monkey Blood (n = 2) IVaIVa IVa Time Iab Formed % IVa Iab Formed % IVa Iab Formed % ‘Va (min)(μM) (μM) Formed* (μM) (μM) Formed* (μM) (μM) Formed* 0 8.0 0.019 0.2412 0.036 0.30 8.5 0.011 0.13 20 8.8 0.056 0.70 12 0.29 2.4 9.2 0.0240.28 40 10 0.072 0.90 11 0.49 4.1 9.2 0.041 0.48 60 10 0.093 1.2 11 0.746.2 8.7 0.048 0.56 *Percentage formed as the starting concentration ofIab.

TABLE 16 Stability of Iab in the Fresh EDTA Blood from Dog (Repeat) andHuman Dog A Blood (n = 2) Dog B Blood (n = 2) Human Blood (n = 2) IVaIVa IVa Time Iab Formed % IVa Iab Formed % IVa Iab Formed % IVa (min)(μM) (μM) Formed* (μM) (μM) Formed* (μM) (μM) Formed* 0 11 0.069 0.63 120.084 0.68 11 0.061 0.55 15 11 0.25 2.3 11 0.25 2.0 11 0.067 0.59 30 110.37 3.4 11 0.43 3.5 7.1 0.057 0.50 45 11 0.53 4.8 13 0.56 4.5 9.8 0.0730.65 60 10 0.67 6.1 14 0.72 5.8 9.8 0.084 0.75 *Percentage formed as thestarting concentration of Iab.

TABLE 17 Stability of Iab in Tris-HCl Buffer Time Tris-HCl (n = 2) (min)Iab (μM) IVa Formed (μM) % IVa Formed* 0 10 0.011 0.11 20 10 0.008 0.08140 7.6 0.008 0.076 60 10 0.009 0.088 *Percentage formed as the startingconcentration of Iab.

To investigate the hydrolysis of tab in the systemic circulation, Iabwas incubated in fresh serum (rat, dog, monkey and human) at 37° C. for90 min. The rate of hydrolysis was the most rapid in the rat serum,followed by dog, monkey and human sera (Table 18). The conversion of Iabto IVa was near stoichiometric.

Serum contains lower ALP activities as compared to tissues (McComb etal., 1979a). In addition, serum also contains ALP isoforms from tissuesources such as bone, liver and intestine, which are attributed toleakage through the blood vessels (Moss, 1983). Therefore, thehydrolysis of Iab in serum was probably mediated by multiple isoforms ofALP.

TABLE 18 Stability of Iab in the Fresh Serum from Rat, Dog, Monkey andHuman Rat Serum Dog Serum Monkey Serum Human Serum (n = 2) (n = 2) (n =2) (n = 2) IVa IVa IVa IVa Time Formed Formed Formed Formed (min) Iab(μM) (μM) Iab (μM) (μM) Iab (μM) (μM) Iab (μM) (μM) 0 6.8 0.25 9.2 0.0669.0 0.20 9.5 0.10 15 5.8 1.1 9.0 0.85 7.3 0.48 9.2 0.10 30 5.4 1.8 7.81.6 6.8 0.75 9.0 0.15 45 4.9 2.8 7.8 2.5 6.5 1.2 9.6 0.20 60 4.3 3.7 7.13.2 6.3 1.5 9.1 0.24 90 3.3 5.4 6.7 4.4 6.3 2.0 9.4 0.32 t_(1/2) 80182** 365** >2000 (min)* *Calculated as the disappearance of Iab. **Thehalf-lives are greater than the incubation period.Hydrolysis of Iab in the Presence of Human Placental ALP

To study the hydrolysis of Iab in a purified form of human ALP, Iab wasincubated in human placental ALP solutions at 10, 100 and 1000 units/Lat 37° C. for 2 hr. The disappearance t_(1/2) of Iab was determined andreported in Table 19. As expected, the rate of hydrolysis was faster inthe solutions with higher ALP activities. IVa was also formedaccordingly (FIG. 8). This indicates that Iab is hydrolyzed by the ALPderived from humans to form IVa.

TABLE 19 Hydrolysis of Iab in Human Placental ALP Solutions ALP Activity(Units/L) (n = 2) 10 100 1000 t_(1/2) (min) 186 14 1.4 Note: Iab wasincubated at a starting concentration of ~10 μM at 37° C. for 120 min.In Vivo StudiesIn Vivo Studies in the Rat

The pharmacokinetic parameters of Iab and IVa in rats after IV and oraladministration of Iab are summarized in Table 20. The plasmaconcentration versus time profiles are shown in FIG. 9. For comparison,the historical data from the pharmacokinetic studies of IVa in rats arealso shown.

The total body clearance (Cl) of Iab following IV administration was 14mL/min/kg, suggesting that Iab is a low clearance compound in rats. Theelimination half-life (t_(1/2)) and mean residence time (MRT) after IVadministration were 0.16 hr and 0.14 hr, respectively. Iab was notdetected beyond 2 hr. The volume of distribution of Iab at steady state(Vss) was 0.12 L/kg, suggesting very limited tissue distribution. Theformation of IVa from Iab after IV administration was rapid; IVa wasdetected at the first sampling time point of 2 min (data not shown). TheIV AUC ratio of IVa formed from Iab vs. from the historical IVa studywas 1.5 (theoretical value for complete conversion=1), suggestingcomplete conversion of Iab to IVa.

With the exception of one sample (5 nM; Table 20), Iab was not detectedin any samples after oral administration. The Tmax of IVa after oraladministration of Iab was 0.83 hr, which is shorter than the historicalTmax of IVa of 2.0 hr, indicating more rapid absorption of IVa followingthe oral administration of the prodrug. The more rapid absorption of IVafrom the prodrug is likely the result of better aqueous solubility oftab as well as rapid hydrolysis of tab to form IVa in the intestine.Although the absolute oral bioavailability of IVa from Iab was 62%,lower than the historical IVa data, the exposure of IVa from the Iab ratoral dose escalation study was superior as compared to the historicaldata with IVa (Table 16 and FIG. 8).

The terminal plasma concentration vs. time profiles of IVa formed fromIab are similar to the historical IVa profiles.

TABLE 20 Pharmacokinetic Parameters of Iab and IVa Following IV and OralAdministration of Iab in Rats (Mean ± SD, n = 3) IVa Formed after DosingPK Parameters Iab with Iab Historical IVa IV Dose (mg/kg) 1.4 free acidor 1 1.1 of IVa equivalent AUC_(tot) (μM · hr)  3.3 ± 1.0  5.4 ± 0.933.3 ± 1.1 CL_(tot) (mL/min/kg)   14 ± 4.2 NA  13 ± 4.0 t_(1/2) (hr) 0.16± 0.052  4.4 ± 1.9 2.4 ± 0.32 MRT (hr) 0.14 ± 0.020 NA 2.2 ± 1.5 Vdss(L/kg) 0.12 ± 0.019 NA 1.5 ± 0.25 IV AUC Ratio of IVa NA  1.5* NA PODose (mg/kg) 7.9 free acid or 5 6.3 of IVa equivalent Tmax (hr) ND 0.83± 0.29  2.0 Cmax (μM) ND**  3.9 ± 0.98 4.5 ± 1.5 C-24 hr (μM) ND ND  9(n =1) AUC^(tot) (μM · hr) ND   13 ± 1.4  15 ± 6.3 t_(1/2) (hr) ND  1.5± 0.24  1.7 ± 0.92 Bioavailability (%) ND 62*** 90 PO AUC Ratio of IVaNA  0.69* NA NA-not applicable; ND-not detected (<5 nM). *The ratioswere calculated from$\frac{{\,^{\prime}043}\mspace{14mu}{AUC}\mspace{14mu}{after}\mspace{14mu}{prodrug}\mspace{14mu}{dosing}}{{\,^{\prime}043}\mspace{14mu}{AUC}\mspace{14mu}{after}\mspace{14mu}{\,^{\prime}043}\mspace{14mu}{dosing}}$for IV and PO, respectively. **Iab was detected in one rat only at 15min (5 nM). ***Calculated from historical IV data of IVa.31. In Vivo Studies in the Dog

The pharmacokinetic parameters of Iab and IVa in dogs after IV and oraladministration of Iab are summarized in Table 21. The plasmaconcentration versus time profiles are shown in FIG. 10. For comparison,the historical data from the pharmacokinetic studies of IVa in dogs arealso shown.

The C1 of Iab after IV administration was 70 mL/min/kg, which issignificantly higher than the liver blood flow of 31 mL/min/kg in dogs,suggestive of potential involvement of extrahepatic hydrolysis and/orother route(s) of elimination (e.g., renal excretion). The t_(1/2) andMRT after IV administration were 0.15 hr and 0.07 hr, respectively. Iabwas not detected beyond 45 min. The Vss of Iab was 0.30 L/kg, suggestinglow potential for tissue distribution. The formation of IVa from Iabafter IV administration was rapid; IVa was detected at the firstsampling time point of 5 min (data not shown). The IV AUC ratio of IVaformed from Iab vs. from the historical IVa study was 0.80, suggestinggood conversion of Iab to IVa.

Iab was detected at 5 nM (LLQ) only at 15 min and 30 min in one dogafter oral administration. The Tmax of IVa after oral administration ofIab was 0.25 hr, which is shorter than the historical Tmax of IVa of 2.9hr, indicating more rapid absorption of IVa following the oraladministration of the prodrug. The absolute oral bioavailability of IVafrom Iab was 94%, higher than the historical IVa data of 57%. Theterminal plasma concentration vs. time profiles of IVa formed from Iabare similar to the historical IVa profiles.

TABLE 21 Pharmacokinetic Parameters of Iab and IVa Following IV and OralAdministration of Iab in Dogs (Mean ± SD, n = 3) IVa Formed after Dosingwith PK Parameters Iab Iab Historical IVa IV Dose (mg/kg) 1.2 free acid1 or 0.95 of IVa equivalent AUC_(tot) (μM · hr) 0.56 ± 0.12   13 ± 2.2 17 ± 3.5 CL_(tot) (mL/min/kg) 70 ± 16  NA  2.4 ± 0.43 t_(1/2) (hr) 0.15± 0.010 5.2 ± 1.0 2.6 ± 1.2 MRT (hr) 0.07 ± 0.006 NA  3.3 ± 0.71 Vdss(L/kg) 0.30 ± 0.094 NA  0.45 ± 0.083 IV AUC Ratio of IVa NA 0.80 NA PODose (mg/kg) 6.6 free acid 5 or 5.2 of IVa equivalent Tmax (hr) ND 0.252.9 ± 1.9 Cmax (μM) ND*  14 ± 1.3 6.5 ± 1.2 C-24 hr (μM) ND 0.26 ± 0.12 0.15 ± 0.093 AUC_(tot) (μM · hr) ND 83 ± 16  47 ± 7.2 t_(1/2) (hr) ND 4.1 ± 0.52  3.8 ± 0.81 Bioavailability (%) ND 94 57 ± 17 PO AUC Ratioof IVa NA 1.7 NA *Iab was detected at 5 nM (LLQ) only at 15 min and 30min in one dog.

To study the effect of food on the oral absorption of IVa afteradministration of Iab and IVa, the dogs were administered either Iab orIVa in capsules under fasting and fed conditions. The study wasconducted in a cross-over fashion, with a one-week washout periodbetween each study. No significant differences in the AUC and Cmax wereobserved between the fasting and fed conditions after administration ofIab (Table 22), whereas a 9-fold improvement in AUC and Cmax wasobserved after administration of IVa with feeding (Table 23). Overall,the effect of feeding was more pronounced in dogs (Table 23) than inhuman subjects (Table 24) receiving IVa. This is suggestive ofquantitative species differences in the effect of food on the oralabsorption of IVa.

The oral absorption of IVa was shown to be dissolution-rate limited inhumans and animal species. The improvement of IVa exposure in humansupon feeding with a high fat meal was presumably due to the increasedsecretion of bile salts which facilitated the dissolution of IVa. Indogs, the lack of the effect of food on the oral absorption of IVa afterIab administration suggests potential benefits in humans, for whom dietmodification may not be required.

TABLE 22 Effect of Food on the Oral Exposure of IVa in Dogs FollowingOral Administration of Iab in Dry-Filled Capsules (20 mg/kg of IVaequivalent) (Mean ± SD, n = 3) Parameters Fasting Fed AUC_(tot) (μM ·hr) 414 ± 70  422 ± 57  Cmax (μM) 70 ± 12 130 ± 102 Tmax (hr) 2.0  1.5 ±0.87 t_(1/2) (hr)  4.2 ± 0.42 3.2 ± 1.0 Note: Iab was detected at <200nM in a few samples at the early time points.

TABLE 23 Effect of Food on the Oral Exposure of IVa in Dogs FollowingOral Administration of IVa in Clinical Form Capsules (20 mg/kg) (Mean ±SD, n = 3) Parameters Fasting Fed Fed/Fasting Ratio AUC_(tot) (μM · hr) 11 ± 1.4  96 ± 10* 9.0 ± 2.2  Cmax (μM)  1.2 ± 0.22   11 ± 0.99* 9.1 ±0.92 Tmax (hr) 3.3 ± 1.2 4.0 t_(1/2) (hr) 5.3 ± 2.7 8.0 ± 3.1*Significantly different from the values under fasting condition.

TABLE 24 Effect of Food on the Oral Exposure of IVa in the First-in-Human Study (Mean ± SD, n = 6) 800 mg 1800 mg Fed/Fasting Fed/FastingFasting Fed Ratio Fasting Fed Ratio AUC_(total) 22 ± 12 53 ± 10 2.4  19± 5.8 82 ± 16 4.3 (μM · hr) Cmax 3.7 ± 1.1 9.8 ± 1.3 2.6  3.3 ± 0.84  15± 5.0 4.5 (μM)

Additional capsule formulation studies were conducted in the same fasteddogs with the clinical and spray dried forms of IVa to compare with Iab.As shown in Table 25, at 20 mg/kg of IVa equivalent, similar exposure ofIVa was observed following administration of Iab and the spray driedform of IVa. However, Iab provided significantly higher exposure of IVaas compared to the clinical form of IVa. The AUC and Cmax of IVa fromthe prodrug were 37 times and 45 times higher, respectively, than thevalues from the clinical form of IVa. The fold increase in dogs islikely an over-prediction of the clinical situation based on theexperience with IVa in development (data not shown).

In the dose escalation study of the spray-dry form of IVa in dogs, theincreases of AUC and Cmax from 20 mg/kg to 75 mg/kg were less thanproportional to dose increase (Table 26). No significant furtherincreases in exposure were observed from 75 mg/kg to 200 mg/kg.

TABLE 25 Oral Exposure of IVa in Dogs After Oral Administration of Iaband Different Formulations of IVa in Capsules (Cross-Over Studies)Parameters Dog#4201 Dog#4202 Dog#4201 Dog#4202 Dog#4201 Dog#4202Formulation Iab Iab IVa IVa spray- ‘IVa ‘IVa spray- dry clinicalclinical dry form form Dose 19 19 20 20 20 20 (mg/kg; ‘IVa eq.)AUC_(tot) 486 514 303* 338 11 16 (μM · hr) Cmax (μM) 80 86 46 81 1.3 2.4Tmax (hr) 1.0 1.0   2.0 1.0 1.0 4.0 t_(1/2) (hr) 4.2 3.4 57 4.2 7.1 4.5*AUC was 0-24 hr.

TABLE 26 Oral Exposure of IVa in Dogs After Oral Administration of theSpray-Dry form of IVa in Capsules Parameters Dog#4201* Dog#4202*Dog#1201 Dog#1202 Dog#2201 Dog#2202 Dose 20 20 75 75 200 200 (mg/kg)AUC_(tot)  303** 338 759 923  935** 1209 (μM · hr) Cmax (μM) 46 81 156192 178 210 Tmax (hr)   2.0 1.0 1.0 1.0    1.0 2.0 t_(1/2) (hr) 57 4.2′2.1 1.9    3.0 2.3 Dose ratio 1:3.8:10  Average 1:2.6:3.4 AUC ratioAverage 1:2.7:3.0 Cmax ratio **As in Table 25. **AUC was 0-24 hr.In Vivo Studies in the Monkey

The pharmacokinetic parameters of Iab and IVa in monkeys after IV andoral administration of Iab are summarized in Table 27. The plasmaconcentration versus time profiles are shown in FIG. 11. For comparison,the historical data from the pharmacokinetic studies of IVa in monkeysare also shown.

The C1 of Iab after IV administration was 4.4 mL/min/kg, suggesting thatIab is a low clearance compound in monkeys. The t_(1/2) and MRT after IVadministration were 1.0 hr and 0.18 hr, respectively. The MRT reflecteda more realistic estimate of the duration of Iab in the plasma since theplasma concentrations of Iab in the terminal phase were low (FIG. 11).Iab was not detected beyond 6 hr. The Vss of Iab was 0.048 L/kg,suggesting very limited tissue distribution. The formation of IVa fromIab after IV administration was rapid; IVa was detected at the firstsampling time point of 5 min (data not shown). The IV AUC ratio of IVaformed from Iab vs. from the historical IVa study was 0.70, suggestinggood conversion of Iab to IVa.

Iab was detected at a concentration of 18 nM at 15 min in only onemonkey after oral administration. The Tmax of IVa after oraladministration of Iab was 0.83 hr, which is shorter than the historicalTmax of IVa of 2.7 hr, indicating more rapid absorption of IVa followingthe oral administration of the prodrug. The absolute oralbioavailability of IVa from Iab was 66%, similar to the historical IVadata of 60%.

The terminal plasma concentration vs. time profiles of IVa formed fromIab are similar to the historical IVa profiles.

TABLE 27 Pharmacokinetic Parameters of Iab and IVa Following IV and OralAdministration of Iab in Monkeys (Mean ± SD, n = 3) IVa Formed afterDosing with PK Parameters Iab Iab Historical IVa IV Dose (mg/kg) 1.3free acid 1 or 1.1 of IVa eqv. AUC_(tot) (μM · hr) 9.5 ± 0.38 7.1 ± 1.09.2 ± 0.53 CL_(tot) (mL/min/kg) 4.4 ± 0.18 NA 4.3 ± 0.26 t_(1/2) (hr)1.0 ± 0.78 4.8 ± 1.9 4.7 ± 0.31 MRT (hr) 0.18 ± 0.059 NA 2.4 ± 0.15 Vdss(L/kg) 0.048 ± 0.017  NA 0.63 ± 0.39  IV AUC Ratio of NA 0.70 NA IVa PODose (mg/kg) 7.1 free acid 5 or 5.6 of IVa eqv. Tmax (hr) ND 0.83 ± 0.142.7 ± 1.2  Cmax (μM) ND*  16 ± 6.4 5.8 ± 1.2  C-24 hr (μM) ND 0.067 ±0.039 0.074 ± 0.032  AUC_(tot) (μM · hr) ND  34 ± 2.7 27 ± 2.9  t_(1/2)(hr) ND 4.8 ± 1.3 4.2 ± 0.64 Bioavailability (%) ND 66 60 ± 4  PO AUCRatio of NA 1.1 NA IVa *Iab was detected at 18 nM at 15 min in only onemonkey.Additional Profiling Section 2Additional Studies with Prodrug Ibb

IV and PO pharmacokinetic studies of Ibb were conducted in rats, dogsand monkeys. In all cases, blood samples were collected in the presenceof EDTA. The presence of EDTA, a known ALP inhibitor, minimizedsignificant ex vivo conversion of Ibb during sample processing. IVb wasrapidly formed following IV administration of Ibb. Good oralbioavailabilities of IVb (50-310%; calculated using the historical IVAUC of IVb) were observed after administration of Ibb in rats, dogs andmonkeys with very low levels of Ibb present in plasma. Since there arehigh levels of ALP expression in the gut, it is likely that followingoral administration of Ibb, Ibb is hydrolyzed by ALP present at thebrush border membranes of the intestinal lumen to form IVb, which israpidly absorbed due to its high permeability.

In vitro incubation studies were conducted for a qualitative assessmentof ALP-dependent hydrolysis of Ibb in different tissues. Ibb washydrolyzed in the presence of serum and hepatocytes from rat, dog,monkey and human, as well as human placental ALP. On the occasions whereIbb and IVb were measured, the conversion of Ibb to IVb was nearstoichiometric. Due to hydrolysis in serum, the protein binding of Ibbcould not be determined. Based on the in vitro data, it is anticipatedthat Ibb will be hydrolyzed by human ALP and that IVb will be formedafter oral administration of Ibb to human subjects.

The crystalline solubility of Ibb-03 at room temperature is >11 mg/mL inthe pH range of 1.5 to 8.7; aqueous solutions containing >75 mg/mL havebeen prepared for in vivo toxicology studies. In comparison, the aqueoussolubility of the parent compound, IVb, at room temperature ascrystalline material was determined to be 0.007-0.19 mg/mL (pH range of1.0-9.6). Ibb-03 exhibits acceptable solution and solid stabilities.

The higher aqueous solubility of Ibb provides a means to overcome thedissolution-rate limited absorption of IVb below certain doses andthereby increased the exposures of IVb in oral dose escalation andtoxicokinetic studies. Ibb, when dosed orally up to 200 mg/kg of IVbbmsequivalent, provided 11- and 2.6-fold (BID) higher AUC of IVb in ratsand dogs, respectively, with relatively low plasma exposure to theprodrug (<0.9 μM), as compared to the AUC from the historical IVbsuspension studies at the similar dose.

Single-Dose Toxicokinetic Study in Sprague Dawley Rats

A 1-day oral toxicokinetic study in rats was conducted by using theprodrug Ibb (monolysine salt). Ibb was administered at dosages of 19,64, and 236 mg/kg (free acid) by oral gavage to three male rats/groupusing water as the vehicle (solution formulation). The dosages ofprodrug free acid correspond to IVb (parent) molar equivalent dosages of15, 51, and 190 mg/kg, respectively. The endpoint evaluated was plasmatoxicokinetics of Ibb and IVb in the individual rats.

The mean toxicokinetic values are provided in Table 28.

TABLE 28 Mean toxicokinetic values for Ibb and IVb in male rats given≦200 mg/kg of Ibb as a single oral dose Dosages (mg/kg) Ibb 19 64 236IVb molar 15 51 190 equiv. Ibb IVb Ibb IVb Ibb IVb Cmax (μM) 0.027  580.14 135 0.25  290 C_(24 h) (μM) <LLQ  0.048 <LLQ  0.29 <LLQ  29 AUC0.030¹ 283² 0.18¹ 997² 0.49¹ 3700² (μM · h) T1/2 (h) 1.3  2.1 4.6  2.13.6   5.8 Values represent means from one to three rats/group for Ibbdata and 3 rats/group for IVb data. LLQ is the lower limit ofquantification. ¹AUC from zero to last time point. ²AUC from zero to ∞

Mean maximum plasma concentration (Cmax) of IVb (parent) was achievedwithin ≈1-3 hours post-dose. In rats given ≦236 mg/kg of Ibb, theincrease in AUC of IVb was nearly proportional with Ibb dosage, and Cmaxincreased in a less than dosage-proportional manner between 19 and 236mg/kg of Ibb. Ibb was detected in plasma of rats given ≧19 mg/kg of Ibbbut at very low concentrations relative to those of IVb.

A comparison of IVb Cmax and AUC obtained in rats given either IVb orIbb, is shown in FIG. 12.

The exposure to IVb is substantially increased following administrationof Ibb compared to that achieved after dosing IVb.

Single-Dose Toxicokinetic and Tolerability Study in Dogs

A two-phase study was conducted to evaluate the tolerability of theprodrug, Ibb (monolysine salt) at dosages of 25, 92, or 250 mg/kg (freeacid, molar equivalent to 20, 74, or 201 mg/kg of parent IVb,respectively) and the toxicokinetics of Ibb and IVb (1). On day 1, Ibbwas administered to one dog/sex/group once daily in dry-filled capsulesat the above dosages. A 1-week washout period was used between doses inthe study. On day 8, Ibb was administered to one dog/sex/group oncedaily as an aqueous solution at 25 mg/kg or twice daily in dry-filledcapsules at 46 or 125 mg/kg BID. The endpoints were: clinical signs,body weight, food consumption, serum chemistry, hematology and plasmatoxicokinetics of Ibb and IVb.

The plasma toxicokinetic values from individual dogs are shown in Table29.

TABLE 29 Toxicokinetic values for IVb in dogs given ≦250 mg/kg of IbbDosages (mg/kg) Ibb 25¹ 92² 250³ IVb molar 20  75  203  equiv. Animal2101M 2201F 3101M 3201F 4101M 4201F Cmax (μM) day 1 72 34  28⁴  66⁴  61⁴ 93⁴ day 8 56 33  74⁴  108⁵  158⁵  88^(4,5) C_(24 h) (μM) day 1 0.0080.688  1.90   0.202   2.61  0.704 day 8 0.547 0.107  10.6   0.307  54.2 1.96 AUC_(0-∞) (μM · h) day 1 259 136 111⁴  329⁴  334⁴ 745⁴ day 8 324139 478⁴ 1053⁵ 1393⁵ 978^(4,5) T½ (h) day 1 1.5 4.5  3.6   2.3   4.1 3.2 day 8 3.3 3.4  5.2   2.4   8.0  3.3 ¹Formulated as dry-filledcapsules on day 1 and as an aqueous solution on day 8 formulated (QDdosing on both days) ²As 92 mg/kg QD on day 1 and as 46 mg/kg BID on day8; formulated as dry-filled capsules on both days ³As 250 mg/kg QD onday 1 and as 125 mg/kg BID on day 8; formulated as dry-filled capsuleson both days ⁴Emesis observed within 2 hours of dosing with capsuleremnant present ⁵Emesis observed within 2 hours of dosing with nocapsule remnant

Low levels (≦0.9 μM) of Ibb were detected in some plasma samples on days1 and 8. Mean maximum plasma concentration (Cmax) of IVb was achievedbetween 1-4 hours post-dose for QD dosing, and 1-2 hours post-seconddose for BID dosing. At 25 mg/kg QD, equivalent Cmax and AUC of IVb wasobserved when Ibb was administered as either dry-filled capsules or anaqueous solution. On day 1, emesis (white, streaked with red thatcontained capsule remnants) was observed at about 0.5-1.25 hours afterdosing in all dogs given ≧92 mg/kg QD (3101M, 3201F, 4101M, and 4201F),which likely contributed to the flat exposure between 92 and 250 mg/kg.On day 8, emesis (white or brown) was observed within 2 hours afterdosing in all dogs given ≧46 mg/kg BID; capsule remnants were onlyobserved in vomitus of two dogs (3101M post-first dose and 4201F postsecond dose). Twice-daily dosing provided greater exposure to IVb indogs than once daily dosing.

Other than emesis, there were no clinical signs observed, and there wereno effects on body weight and food consumption.

Despite the emesis observed in dogs, a higher IVb AUC is observed indogs given Ibb than that in dogs given IVb. A comparison of IVb AUCobtained in dogs given either Ibb or IVb (2), is shown in FIG. 13.

The absolute oral bioavailability of IVb, parent compound, afteradministration of aqueous solutions of Ibb-03, the phosphate prodrug,ranged from 50% to 310% in rats, dogs, and monkeys. These calculationsare based on historical IV data. The exposure of IVb, after oraladministration of aqueous solutions of the prodrug, Ibb-03, dosed up to190 mg/kg of IVb equivalent is 3-10 fold higher in the rat oral doseescalation study as compared to the historical data with IVb. WhenIbb-03 is dosed BID as drug in capsule in dogs, 2-3 fold improvement inexposure is achieved compared to historical data from pre-ECN toxicologystudies with BID dosing of IVb as suspensions. The in vivo exposure datafrom drug in capsule formulations suggests that oral exposure of IVb inhumans could be significantly improved by administration of traditionalsolid oral dosage forms of Ibb.

Ibb is the phosphate prodrug of the N-hydroxymethyl adduct of IVb and ishydrolyzed by alkaline phosphatase (ALP) to form IVb. Afteradministration of Ibb to animals, therefore, plasma samples wereprepared from blood collected in the presence of EDTA, a known ALPinhibitor. Conversion of Ibb to IVb was minimal (<2%) in the bloodcontaining EDTA (rat, dog, monkey and human). No significant ex vivoconversion of Ibb is expected during sample storage (−20° C.) andanalysis of Ibb.

The hydrolysis of Ibb was studied in animal and human in vitro systems.Since multiple ALP isoforms are widely distributed in various tissues,quantitative in vitro to in vivo correlations were not attempted(Fishman et al., 1968; Komoda et al., 1981; Moss, 1983; Yora andSakagishi, 1986; Sumikawa et al., 1990). Therefore, the studies werelimited to a qualitative assessment of ALP-dependent hydrolysis indifferent tissues. Ibb was hydrolyzed in the presence of serum (rat,dog, monkey and human), hepatocytes (rat, dog and human) as well as inhuman placental ALP. No turnover was observed in monkey hepatocytes. Theconversion of Ibb to IVb was near stoichiometric. Due to hydrolysis inserum, the protein binding of Ibb could not be determined.

Ibb was completely hydrolyzed to form IVb in Caco-2 cells and, asexpected, very low levels of Ibb were detected in rat, dog and monkeyplasma after oral administration of Ibb. These data are consistent withreports describing the high levels of ALP expression in the gut (McCombet al., 1979a; Butterworth, 1983). The membrane-bound ALP is mostlylocalized in the brush border membranes of the microvilli lining theintestinal lumen (Butterworth, 1983; Testa and Mayer, 2003). It islikely that following oral administration of Ibb, Ibb is hydrolyzed byALP present at the brush border membranes of the intestinal lumen toform IVb, which is rapidly absorbed due to its high permeability.

Although different isoforms of ALP exist in different tissues andspecies, the substrate specificity for ALP is relatively broad (Heimbachet al., 2003), which is consistent with the above findings for Ibb.

IVb was rapidly formed following IV administration of Ibb in rats, dogsand monkeys. The IV AUC conversion ratios were 0.62 in rats, 1.6 in dogsand 1.7 in monkeys, suggesting satisfactory to good conversion from Ibbto IVb.

Good oral bioavailabilities (50-310%; calculated using the historical IVAUC of IVb) of IVb were observed after administration of Ibb in rats,dogs and monkeys. More significantly, the higher aqueous solubility ofIbb lessened the dissolution-rate limited absorption of IVb and therebyincreased the exposures of IVb in oral dose escalation and toxicokineticstudies as compared to the exposures from the historical IVb studies(Discovery Toxicology section Tables 28-29 and FIG. 12-13).

Methods

The studies described in this report used the mono-lysine salt of Ibb.

Quantitation of Ibb and IVb by LC/MS/MS

An LC/MS/MS method was developed for the analysis of Ibb and IVb inplasma samples from the animal pharmacokinetic studies as well as inacetonitrile supernatant from in vitro incubation studies. For theanalysis in plasma, a Packard Multiprobe instrument was used to transfer50 μL of each standard, QC, and plasma sample to a clean 96-well platefor protein precipitation extraction. After the addition of 200 μL ofacetonitrile containing the internal standard IVc, the samples werevortex mixed and the resulting supernatant was separated from theprecipitated proteins by centrifugation for 10 min. For the analysis inthe supernatant generated from the in vitro studies, an equal volume ofthe supernatant and acetonitrile containing the internal standard wasmixed. An aliquot of the above supernatant was transferred using aTomtec automated liquid handler to a second clean 96-well plate. Anequal volume of water was added, and the plate was capped and vortexmixed.

The HPLC system consisted of Shimadzu LC10ADvp pumps (Columbia, Md.) anda HTC PAL autosampler (Leap Technologies, Cary, N.C.) linked to aPhenomenex Synergi Fusion-RP analytical column (2.0×50 mm, 5μ; Torrance,Calif.). Mobile phase A consisted of 5 mM ammonium formate in water;mobile phase B was 100% acetonitrile. LC flow rate was 0.38 mL/min. Theinitial mobile phase composition was 2% B, ramped to 50% B over 1.8 minand held for 0.5 min, ramped to 100% B over 0.1 min and held for 0.5min, returned to initial conditions over the next 0.1 min, andre-equilibrated. Total analysis time was 4.0 min. The retention time forIbb, IVb and IVc was 1.42, 2.21 and 1.73 min, respectively.

The HPLC system was interfaced to a Sciex API4000 triple quadrupole massspectrometer (Toronto, Canada) equipped with the Turboionspray sourceset at 550° C. and the ionspray voltage set to 4.5 kV. UHP nitrogen wasused as nebulizer and auxiliary gas with the pressure of 80 psi and 7L/min, respectively. The collision energies for Ibb, IVb and IVc were19, 25 and 29 volts, respectively. Data acquisition utilized selectedreaction monitoring (SRM). Ions representing the positive ion mode(M+H)⁺ species for Ibb, IVb and the internal standard were selected inMS 1 and collisionally dissociated with nitrogen and optimized collisionenergies to form specific product ions subsequently monitored by MS2.The SRM transitions for Ibb, IVb and IVc were m/z 558→432, 448→202 and474→256, respectively.

Standard curves ranging from 5 nM to 10 μM were prepared from stocksolutions and serially diluted in matrix for both Ibb and IVb. Standardcurves were aliquoted in duplicate, extracted with the samples, andinjected at the beginning, middle, and end of the analytical sequence.The standard curves were fitted with a linear regression weighted byreciprocal concentration 1/x². Data and chromatographic peaks wereprocessed and concentrations of standards and unknowns were quantitatedusing PEBiosystems Analyst™ 1.1.

In Vitro Methods

(1) Stability of Ibb in EDTA Blood, Serum and Tris-HCl Buffer

The stability of Ibb was studied in fresh blood and serum from rat, dog,monkey and human (n=2). The blood was collected in vacutainerscontaining K₂EDTA (Becton Dickinson, Franklin Lakes, N.J.). The serumwas collected in vacutainers containing no anticoagulant. Ibb wasincubated at a starting concentration of approximately 15 μM for 90 minat 37° C. Serial samples were taken at the pre-determined times.Aliquots of blood samples (200 μL) were first mixed with 100 μL of waterfollowed by 400 μL of acetonitrile. The serum samples (50 μL) were addedinto microtainers containing K₂EDTA (Becton Dickinson, Franklin Lakes,N.J.) followed by the addition of 100 μL of acetonitrile. Thesupernatant was analyzed for both Ibb and IVb by LC/MS/MS.

The stability of Ibb was also evaluated, as described above, in Tris-HClbuffer (0.1 M, pH 7.5).

(2) Hydrolysis of Ibb in the Presence of Human Placental ALP

Solid human placental ALP was obtained from Sigma (P-3895, St. Louis,Mo.). A solution of 1000 units/L was prepared in Tris-HCl buffer (0.1 M,pH 7.5). Solutions of 100 and 10 units/L were obtained by serialdilution. Ibb was incubated in the 10, 100 and 1000 units/L solutions(n=2) at 37° C. for 2 hr. The starting concentration of Ibb in theincubation was 10 μM. Aliquots of 100 μL samples were taken atpre-determined times and added into K₂EDTA microtainers followed by theaddition of 200 μL of acetonitrile. The supernatant was analyzed forboth Ibb and IVb by LC/MS/MS.

In Vivo Studies

All blood samples (0.3 mL) were collected in microtainers containingK₂EDTA (Becton Dickinson, Franklin Lakes, N.J.) and placed on chippedice. After centrifugation, plasma was separated and stored at −20° C.until analysis. The mono-lysine salt of Ibb (Form 3) was used for thepharmacokinetic studies. The dosing solutions of Ibb were prepared ineither sterile water (for IV administration in dogs and monkeys) ordistilled water (for all other dose administration).

(1) In Vivo Studies in the Rat

Male Sprague-Dawley rats (300-350 g, Hilltop Lab Animals, Inc.,Scottsdale, Pa.) with cannulas implanted in the jugular vein were used.The rats were fasted overnight in the PO pharmacokinetic studies. Bloodsamples were collected from the jugular vein.

In the IV study, Ibb was delivered at 1.3 mg/kg (free acid, or 1.0 mg/kgof IVb equivalent) as a bolus over 0.5 min (n=3). The concentration ofthe dosing solution was 1.3 mg/mL, and the dosing volume was 1 mL/kg.Serial blood samples were collected before dosing and at 2, 10, 15, 30,45 min, 1, 2, 4, 6, 8 and 24 hr after dosing.

In the PO study, Ibb was administered at 6.6 mg/kg (free acid, or 5.2mg/kg of IVb equivalent) by oral gavage (n=3). The concentration of thedosing solution was 1.3 mg/mL, and the dosing volume was 5 mL/kg. Serialblood samples were collected before dosing and at 15, 30, 45 min, 1, 2,4, 6, 8 and 24 hr after dosing.

(2) In Vivo Studies in the Dog

The IV and PO studies of Ibb were conducted in male beagle dogs (10±0.78kg, Marshall Farms USA Inc., North Rose, N.Y.). Three dogs were used ineach study. Of the three dogs, two same dogs were used for both IV andPO studies. There was a two-week washout period between the IV and POstudies.

In the IV study, Ibb was infused via the cephalic vein at 1.3 mg/kg(free acid, or 1.0 mg/kg of IVb equivalent) over 5 min at a constantrate of 0.1 mL/kg/min. The concentration of the dosing solution was 2.6mg/mL, and the dosing volume was 0.5 mL/kg. Serial blood samples werecollected from the femoral artery before dosing and at 5, 10, 15, 30, 45min, 1, 2, 4, 6, 8 and 24 hr after dosing.

In the PO study, the dogs were fasted overnight before dosing. Ibb wasadministered by oral gavage at 7.0 mg/kg (free acid, or 5.6 mg/kg of IVbequivalent). The concentration of the dosing solution was 7.0 mg/mL, andthe dosing volume was 1 mL/kg. Serial blood samples were collectedbefore dosing and at 15, 30, 45 min, 1, 2, 4, 6, 8 and 24 hr afterdosing.

(3) In Vivo Studies in the Monkey

The IV and PO studies of Ibb were conducted in a crossover fashion inthree male cynomolgus monkeys (9.9±2.4 kg, Charles River BiomedicalResearch Foundation, Houston, Tex.). There was a two-week washout periodbetween the IV and PO studies.

In the IV study, Ibb was infused via the femoral vein at 1.3 mg/kg (freeacid, or 1.1 mg/kg of IVb equivalent) over 5 min at a constant rate of0.1 mL/kg/min. The concentration of the dosing solution was 2.7 mg/mL,and the dosing volume was 0.5 mL/kg. Serial blood samples were collectedfrom the femoral artery before dosing and at 5, 10, 15, 30, 45 min, 1,2, 4, 6, 8 and 24 hr after dosing.

In the PO study, the monkeys were fasted overnight before dosing. Ibbwas administered by oral gavage at 5.8 mg/kg (free acid, or 4.7 mg/kg ofIVb equivalent). The concentration of the dosing solution was 5.8 mg/mL,and the dosing volume was 1 mL/kg. Serial blood samples were collectedbefore dosing and at 15, 30, 45 min, 1, 2, 4, 6, 8 and 24 hr afterdosing.

(4) Data Analysis

All results are expressed as mean±SD, unless specified otherwise.

The pharmacokinetic parameters of Ibb and IVb were calculated byNon-Compartmental Analysis using the KINETICAT™ software program(version 4.0.2, InnaPhase Co., Philadelphia, Pa.). The Cmax and Tmaxvalues were recorded directly from experimental observations. TheAUC_(0-n) and AUC_(tot) values were calculated using the mixedlog-linear trapezoidal summations. The total body clearance (Cl), meanresidence time (MRT), and the steady state volume of distribution (Vss)were also calculated after intravenous administration. The absolute oralbioavailability (expressed as %) was estimated by taking the ratio ofdose-normalized AUC values after oral doses to those after intravenousdoses.

The in vitro intrinsic clearance of Ibb in hepatocytes (Cl_(int)) wascalculated as follows:Cl_(int)(μL/min/million cells)=Rate/C _(E)where Rate is the rate of metabolism in hepatocytes (pmol/min/millioncells), and C_(E) is the concentration of Ibb in the incubation.

The in vivo intrinsic hepatic clearance of Ibb (Cl_(int,in vivo)) wascalculated as follows:

${C\;{l_{{{int} \cdot {in}}\mspace{14mu}{vivo}}\left( {{mL}\text{/}\min\text{/}{kg}} \right)}} = {{Cl}_{int} \times \frac{120\mspace{14mu}\left( {{million}\mspace{14mu}{cells}} \right)}{g\mspace{14mu}{liver}} \times \frac{\chi\mspace{14mu} g\mspace{14mu}{liver}}{{kg}\mspace{14mu}{body}\mspace{14mu}{weight}} \times \frac{1}{1000}}$where χ is 40, 32, 30 and 26 g liver/kg body weight for the rat, dog,monkey and human, respectively (Davis and Morris, 1993).

The hepatic clearance Cl_(H) was calculated from the following equationusing the well-stirred model:

${C\;{l_{H}\left( {{mL}\text{/}\min\text{/}{kg}} \right)}} = \frac{{Qh} \times {Cl}_{{{int} \cdot {in}}\mspace{14mu}{vivo}}}{{Qh} + {Cl}_{{{int} \cdot {in}}\mspace{14mu}{vivo}}}$where Qh is the liver blood flow of 55, 31, 44 and 21 mL/min/kg for therat, dog, monkey and human, respectively (Davis and Morris, 1993).

The hepatic extraction ration (ER) was calculated at follows:ER=Cl_(H) /QhStudent's t-test was used for statistical analysis (Microsoft® Excel,Redmond, Wash.). Differences were considered statistically significantat the level of P<0.05.In Vitro StudiesStability of Ibb in EDTA Blood, Serum and Tris-HCl Buffer

As part of the analytical assay validation, the stability of Ibb wasstudied in blood containing EDTA, which is known to be an inhibitor ofALP (Bowers, Jr. and McComb, 1966; Yora and Sakagishi, 1986). Afterincubation at 37° C. for 90 min, there was less than 2% conversion ofIbb to IVb in blood containing EDTA and in the presence of Tris-HClbuffer (Tables 30 and 31). The above small percentages of conversionobserved at 37° C. indicate that conversion under the sample storageconditions (−20° C.) is unlikely. Therefore, relatively minimal ex vivoconversion to IVb is expected during the analysis of Ibb.

TABLE 30 Stability of Ibb in the Fresh EDTA Blood from Rat, Dog andMonkey Rat Blood Dog Blood Monkey Blood (n = 2) (n = 2) (n = 2) IVb IVbIVb Time Ibb Formed % IVb Ibb Formed % IVb Ibb Formed % IVb (min) (μM)(μM) Formed* (μM) (μM) Formed* (μM) (μM) Formed* 0 15 0.23 1.6 13 0.0890.68 16 0.075 0.47 15 15 0.22 1.5 12 0.079 0.60 19 0.12 0.76 30 18 0.261.7 15 0.085 0.65 18 0.13 0.80 45 15 0.29 1.9 16 0.095 0.73 18 0.13 0.8260 16 0.31 2.0 13 0.088 0.67 19 0.14 0.86 90 17 0.32 2.1 16 0.11 0.82 170.14 0.89 *Percentage formed as the starting concentration of Ibb.

TABLE 31 Stability of Ibb in the Fresh EDTA Blood from Human, andTris-HCl Buffer Human Blood Tris-HCl Buffer (n = 2) (n = 2) IVb IVb TimeFormed % IVb Formed % IVb (min) Ibb (μM) (μM) Formed* Ibb (μM) (μM)Formed* 0 16 0.099 0.62 14 0.30 2.2 15 15 0.093 0.58 12 0.30 2.1 30 160.097 0.60 13 0.33 2.3 45 16 0.11 0.67 12 0.41 2.9 60 16 0.10 0.65 130.51 3.7 90 18 0.12 0.73 12 0.51 3.6 *Percentage formed as the startingconcentration of Ibb.

To investigate the hydrolysis of Ibb in the systemic circulation, Ibbwas incubated in fresh serum (rat, dog, monkey and human) at 37° C. for90 min. The rate of hydrolysis was most rapid in the monkey serum,followed by human, dog and rat sera (Table 3). The conversion of Ibb toIVb was near stoichiometric.

Serum contains lower ALP activities as compared to tissues (McComb etal., 1979a). In addition, serum also contains ALP isoforms from tissuesources such as bone, liver and intestine, as a result of enzyme leakagethrough the blood vessels (Moss, 1983). Therefore, the hydrolysis of Ibbin serum was probably mediated by multiple isoforms of ALP.

TABLE 32 Stability of Ibb in the Fresh Serum from Rat, Dog, Monkey andHuman Monkey Rat Serum Dog Serum Serum Human Serum (n = 2) (n = 2) (n =2) (n = 2) IVb IVb IVb IVb Formed Ibb Formed Ibb Formed Ibb Formed Time(min) Ibb (μM) (μM) (μM) (μM) (μM) (μM) (μM) (μM)  0 12 0.095 13 0.13 140.34 13 0.14 15 9.4 0.33 12 0.66 6.6 6.5 9.0 0.96 30 9.2 0.60 11 1.2 3.18.5 10 2.0 45 9.4 0.92 10 1.7 1.7 10 10 3.1 60 9.3 1.3 10 2.4 0.85 118.0 3.6 90 9.0 2.2   8.9 3.6 0.22 13 6.5 5.0 t_(1/2) (min)* 704** 167**15 75 *Calculated as the disappearance of Ibb. **The half-lives aregreater than the incubation period.Hydrolysis of Ibb in the Presence of Human Placental ALP

To study the hydrolysis of Ibb in a purified form of human ALP, Ibb wasincubated at 37° C. (2 hr) with solutions containing human placental ALP(10, 100 and 1000 units/L). The disappearance t_(1/2) of Ibb wasdetermined (Table 4). As expected, the rate of hydrolysis was faster inthe solutions with higher ALP activities. IVb was also formedaccordingly (FIG. 14). This indicates that Ibb is hydrolyzed by the ALPderived from humans to form IVb.

TABLE 33 Hydrolysis of Ibb in Human Placental ALP Solutions ALP Activity(Units/L) (n = 2) 10 100 1000 t_(1/2) (min) 198 16 2.0 Note: Ibb wasincubated at a starting concentration of 10 μM at 37° C. for 120 min.In Vivo StudiesIn Vivo Studies in the Rat

The pharmacokinetic parameters of Ibb and IVb in rats after IV and oraladministration of Ibb are summarized in Table 34. The plasmaconcentration versus time profiles are shown in FIG. 15. For comparison,the historical data from the pharmacokinetic studies of IVb in rats arealso shown.

The total body clearance (Cl) of Ibb following IV administration was 19mL/min/kg, suggesting that Ibb is a low to moderate clearance compoundin rats. The elimination half-life (t_(1/2)) and mean residence time(MRT) after IV administration were 0.18 hr and 0.079 hr, respectively.Iab was not detected beyond 2 hr. The volume of distribution of Ibb atsteady state (Vss) was 0.10 L/kg, suggesting very limited tissuedistribution. The formation of IVb from Ibb after IV administration wasrapid; IVb was detected at the first sampling time point of 2 min (datanot shown). The IV AUC ratio of IVb formed from Ibb vs. from thehistorical IVb study was 0.62 (theoretical value for completeconversion=1), indicating satisfactory conversion of Ibb to IVb in ratsafter IV dosing.

Ibb was detected (<10 nM) in the plasma (0.25 and 0.5 hr) after oraladministration. The Tmax of IVb after oral administration of Ibb was0.83 hr, which is shorter than the historical Tmax of IVb of 4.7 hr,indicating more rapid absorption of IVb following the oraladministration of the prodrug. The more rapid absorption of IVb from theprodrug is likely the result of better aqueous solubility of Ibb as wellas rapid hydrolysis of Ibb to form IVb in the intestine. The absoluteoral bioavailability of IVb from Ibb was 50%, similar to the historicalIVb value of 60% (Table 34). Moreover, the exposure of IVb from the Ibbrat oral dose escalation study was superior as compared to thehistorical data with IVb (Table 31 and FIG. 14).

TABLE 34 Pharmacokinetic Parameters of Ibb and IVb Following IV and OralAdministration of Ibb in the Rat (Mean ± SD, n = 3) IVb Formed Ibb afterDosing with PK Parameters (03-002) Ibb Historical IVb IV Dose (mg/kg)1.3 free acid 1 or 1.0 of IVb eqv. AUC_(tot) (μM * hr) 2.5 ± 1.4 15 ±5.4 24 ± 3.2 CL_(tot) (mL/min/kg)  19 ± 8.9 NA  1.6 ± 0.20 T_(1/2) (hr) 0.18 ± 0.050 3.0 ± 1.8  5.9 ± 4.9 MRT (hr) 0.079 ± 0.041 NA 5.6 ± 3.6Vdss (L/kg)  0.10 ± 0.093 NA 0.49 ± 0.26 IV IVb AUC NA 0.62* NA Ratio PODose (mg/kg) 6.6 free acid 5 or 5.2 of IVb eqv. Tmax (hr) 0.25 0.83 ±0.14  4.7 ± 1.2 Cmax (μM) 0.008 ± 0.003 19 ± 1.8 9.5 ± 2.8 C-24 hr (μM)ND 0.009 ± 0.004  0.16 (n = 2) AUC_(tot) (μM * hr) ND 62 ± 3.3 86 ± 33T_(1/2) (hr) ND  2.2 ± 0.11  3.7 ± 0.86 Bioavailability ND 50**   60 (%) PO IVb AUC NA 0.69* NA Ratio NA—not applicable; ND—not detected (<5nM). *The ratios were calculated from IVb AUC after prodrug dosing/IVbAUC after IVb dosing for IV and PO, respectively. **Calculated fromhistorical IV data of IVb.In Vivo Studies in the Dog

The pharmacokinetic parameters of Ibb and IVb in dogs after IV and oraladministration of Ibb are summarized in Table 35. The plasmaconcentration versus time profiles are shown in FIG. 16. For comparison,the historical data from the pharmacokinetic studies of IVb in dogs arealso shown.

The Cl of Ibb after IV administration was 27 mL/min/kg, similar to theliver blood flow of 31 mL/min/kg in dogs, suggesting that Ibb is a highclearance compound in dogs. The t_(1/2) and MRT after IV administrationwere 0.83 hr and 0.21 hr, respectively. The MRT reflected a morerealistic estimate of the duration of Ibb in the plasma since the plasmaconcentrations of Ibb in the terminal phase were low (FIG. 3). Ibb wasnot detected beyond 4 hr. The Vss of Ibb was 0.35 L/kg, suggestinglimited tissue distribution. The formation of IVb from Ibb after IVadministration was rapid; IVb was detected at the first sampling timepoint of 5 min (data not shown). The IV AUC ratio of IVb formed from Ibbvs. from the historical IVb study was 1.6, suggesting completeconversion of Ibb to IVb in dogs after IV administration.

Ibb was detected (Cmax=0.034 nM) in plasma samples at early time points(up to 2 hr in one dog) following oral administration. The Tmax of IVbafter oral administration of Ibb was 0.40 hr, similar to the historicalTmax of IVb of 0.50 hr. The absolute oral bioavailability of IVb fromIbb was 310%, similar to the historical IVb data of 179%. Moreover, theexposure of IVb from the Ibb dog tolerability study (dose escalation)was greater when compared to the historical data with IVb (Table 31 andFIG. 15).

The terminal plasma concentration vs. time profiles of IVb formed fromIbb are similar to the historical IVb profiles (FIG. 16).

TABLE 35 Pharmacokinetic Parameters of Ibb and IVb Following IV and OralAdministration of Ibb in the Dog (Mean ± SD, n = 3) IVb Formed afterDosing with PK Parameters Ibb Ibb Historical IVb IV Dose (mg/kg) 1.3free acid (03-001) 1 (n = 2) or 1.0 of IVb eqv. AUC_(tot) (μM * hr)  1.4± 0.18 6.2 ± 0.80 3.8 CL_(tot) (mL/min/kg)  27 ± 3.2 NA 9.8 T_(1/2) (hr)0.83 ± 0.58 2.0 ± 0.21 1.6 MRT (hr)  0.21 ± 0.043 NA 1.8 Vdss (L/kg) 0.35 ± 0.091 NA 1.1 IV IVb AUC NA 1.6* NA Ratio PO Dose (mg/kg) 7.0free acid (03-002) 5 (n = 3) or 5.6 of IVb eqv. Tmax (hr) 0.33 ± 0.140.40 ± 0.13  0.50 ± 0.25 Cmax (μM) 0.034 ± 0.018 20 ± 2.4   7.7 ± 0.71C-24 hr (μM) ND 0.037 ± 0.026  0.034 (n = 2)    AUC_(tot) (μM * hr)0.059 (n = 1) 66 ± 17  30 ± 11 T_(1/2) (hr) NA 2.6 ± 0.21  2.7 ± 0.40Bioavailability NA 310**   179 ± 56  (%) PO IVb AUC NA 2.2* NA Ratio*The ratios were calculated from IVb AUC after prodrug dosing/IVb afterIVb dosing for IV and PO, respectively. **Calculated from historical IVdata of IVbIn Vivo Studies in the Monkey

The pharmacokinetic parameters of Ibb and IVb in monkeys following IVand oral administration of Ibb are summarized in Table 36. The plasmaconcentration versus time profiles are shown in FIG. 17. For comparison,the historical data from the pharmacokinetic studies of IVb in monkeysare also shown.

The Cl of Ibb after IV administration was 28 mL/min/kg, suggesting thatIbb is a moderate to high clearance compound in monkeys. The t_(1/2) andMRT after IV administration were 0.10 hr and 0.093 hr, respectively. TheVss of Ibb was 0.15 L/kg, suggesting very limited tissue distribution.The formation of IVb from Ibb after IV administration was rapid; IVb wasdetected at the first sampling time point of 5 min (data not shown). TheIV AUC ratio of IVb formed from Ibb vs. from the historical IVb studywas 1.7, suggesting complete conversion of Ibb to IVb in monkeys afterIV dosing.

Ibb was not detected (LLQ=5 nM) in any plasma samples after oraladministration. The Tmax of IVb after oral administration of Ibb was 1.5hr, similar to the historical Tmax of IVb of 2.5 hr. The absolute oralbioavailability of IVb from Ibb was 187%, which is higher that thehistorical IVb data of 49% (Table 36).

The terminal plasma concentration vs. time profiles of IVb formed fromIbb are similar to the historical IVb profiles (FIG. 17).

TABLE 36 Pharmacokinetic Parameters of Ibb and IVb Following IV and OralAdministration of Ibb in the Monkey (Mean ± SD, n = 3) IVb Formed afterDosing with PK Parameters Ibb Ibb Historical IVb IV Dose (mg/kg) 1.3free acid 1 (n = 3) or 1.1 of IVb eqv. AUC_(tot) (μM * hr) 1.5 ± 0.40 19 ± 1.8  10 ± 1.2 CL_(tot) 28 ± 6.8  NA  3.7 ± 0.43 (mL/min/kg)T_(1/2) (hr) 0.10 ± 0.042 6.5 ± 2.4 19 ± 20 MRT (hr)  0.093 ± 0.00076 NA5.6 ± 6.3 Vdss (L/kg) 0.15 ± 0.039 NA 1.2 ± 1.4 IV IVb AUC NA 1.7* NARatio PO Dose (mg/kg) 5.8 free acid 5 (n = 2) or 4.7 of IVb eqv. Tmax(hr) NA  1.5 ± 0.87 2.5 Cmax (μM) ND 31 ± 11 4.2 C-24 hr (μM) ND  0.11 ±0.075 0.24 AUC_(tot) (μM * hr) ND  88 ± 4.6 24 T_(1/2) (hr) NA 4.1 ± 1.111 Bioavailability NA 187**   49 (%) PO IVb AUC NA 3.4* NA Ratio *Theratios were calculated from IVb AUC after prodrug dosing/IVb for AUCafter IVb dosing IV and PO, respectively. **Calculated from historicalIV data of IVbProfiling Section 3:Additional Studies with Prodrug IcbSingle-Dose Toxicokinetic Tolerability Study in CD Rats

A 1-day oral toxicokinetic study in rats was conducted using the prodrugIcb (disodium salt). Icb was administered at dosages of 5, 25, and 200mg/kg (free acid) by oral gavage to three male rats/group using water asthe vehicle (solution formulation). The dosages of prodrug free acidcorrespond to IVc (parent) molar equivalent dosages of 4.5, 21, and 163mg/kg, respectively. The endpoint evaluated was plasma toxicokinetics ofIcb and IVc in the individual rats.

The mean toxicokinetic values are provided in Table 37.

TABLE 37 Mean toxicokinetic values for Icb and IVc in male rats given≦200 mg/kg of Icb as a single oral dose Dosages (mg/kg) Icb 5 25 200 IVcmolar 4.5 21 163 equiv. IVc IVc IVc Cmax (μM) 29   98 281 C_(24h) (μM) 0.029    0.35  58 AUC 109¹    586¹  2925²  (μM · h) T½ (h) 3.2   2.3   3.4 Values represent means from three rats/group for Icb data and 3rats/group for IVc data. ¹AUC from zero to ∞ ²AUC from zero to 24 h

Mean maximum plasma concentration (Cmax) of IVc (parent) was achievedwithin ≈1.7 hour post-dose. In rats given ≦267 mg/kg of Icb, theincrease in AUC of IVc was nearly proportional with Icb dosage, and Cmaxincreased in a less than dosage-proportional manner between 25 and 200mg/kg of Icb. Icb was not detected in plasma of rats given ≦25 mg/kg ofIcb, and very low concentrations (≈0.02-0.04 μM) were detected in plasmaof rats given 200 mg/kg of Icb at a few time points.

A comparison of IVc AUC obtained in rats given either IVc (1) or Icb, isshown in FIG. 18.

The AUC of IVc is similar after administration of either parent orprodrug at lower dosages (e.g., ≦25 mg/kg) because both can beformulated as solutions (PEG-400/ethanol/0.1N NaOH for parent and waterfor prodrug) but at a high dosage (e.g., 200 mg/kg) the neutral parentcan only be formulated as a suspension whereas the prodrug salt can beformulated as an aqueous solution, which provides superior exposure ofIVc.

Single-Dose Toxicokinetic and Tolerability Study in Dogs

A two-phase study was conducted to evaluate the tolerability of theprodrug, Icb (monotromethamine salt) at dosages of 25, 92, or 250 mg/kg(free acid, molar equivalent to 20, 75, or 203 mg/kg of parent IVc,respectively) and the toxicokinetics of Icb and IVc (2). On day 1, Icbwas administered to one dog/sex/group once daily in dry-filled capsulesat the above dosages. A 1-week washout period was used between doses inthe study. On day 8, Icb was administered to one dog/sex/group oncedaily as an aqueous solution at 25 mg/kg or twice daily in dry-filledcapsules at 46 or 125 mg/kg BID. The endpoints were: clinical signs,body weight, food consumption, serum chemistry, hematology and plasmatoxicokinetics of Icb and IVc.

The plasma toxicokinetic values from individual dogs are shown in Table38.

TABLE 38 Toxicokinetic values for IVc in dogs given ≦250 mg/kg of IcbDosages (mg/kg) Icb 25¹ 92² 250³ IVc molar 20  75  203  equiv. Animal2101M 2201F 3101M 3201F 4101M 4201F Cmax (μM) day 1 65.1 24.6  117⁴ 157 92.6⁴  88.9⁴ day 8 59.4 46.8  121 104  226⁵  96.9⁴ C_(24 h) (μM) day 10.008 0.688   1.90  0.202   2.61  0.704 day 8 0.547 0.107  10.6  0.307 54.2  1.96 AUC_(0-∞) (μM · h) day 1 365 122  820⁴ 730  739⁴ 523⁴ day 8362 173 1137 490 3783⁵ 596^(4,5) T½ (h) day 1 1.5 4.5   3.6  2.3   4.1 3.2 day 8 3.3 3.4   5.2  2.4   8.0  3.3 ¹Formulated as dry-filledcapsules on day 1 and as an aqueous solution on day 8 formulated (QDdosing on both days) ²As 92 mg/kg QD on day 1 and as 46 mg/kg BID on day8; formulated as dry-filled capsules on both days ³As 250 mg/kg QD onday 1 and as 125 mg/kg BID on day 8; formulated as dry-filled capsuleson both days ⁴Emesis observed within 2 hours of dosing with capsuleremnant present ⁵Emesis observed within 2 hours of dosing with nocapsule remnant

Low levels (≦0.1 μM) of Icb were detected in some plasma samples on days1 and 8. Mean maximum plasma concentration (Cmax) of IVc was achievedbetween 1-2 hours post-dose for QD dosing, and 1-2 hours post-seconddose for BID dosing. At 25 mg/kg QD, equivalent Cmax and AUC of IVc wasobserved when Icb was administered as either dry-filled capsules or anaqueous solution. On day 1, emesis (white or brown, streaked with redthat contained capsule remnants) was observed at about 1-1.25 hoursafter dosing in dogs given ≧92 mg/kg QD (3101M, 4101M, and 4201F), whichlikely contributed to the flat exposure between 92 and 250 mg/kg. On day8, emesis was observed within 2 hours after dosing in both dogs given125 mg/kg BID but with different results Animal 4101M had substantialexposure despite emesis, consistent with the absence of capsule remnantin the vomitus.

Only low levels (≦0.1 μM) of Icb were detected in some plasma samples ondays 1 and 8.

Other than emesis, there were no clinical signs observed, and there wereno effects on body weight and food consumption.

Despite the emesis observed in dogs, a higher IVc AUC is observed indogs given Icb than that in dogs given IVc. A comparison of IVc AUCobtained in dogs given either Icb or IVc (3, 4), is shown in FIG. 19.

Vehicles and Formulations

Summary of Formulations Used for Key PK and Safety Studies

All in vivo PK studies in rats, dogs, and monkeys were performed usingaqueous solutions for PO and IV dosing. Pre-ECN toxicology studies indogs were performed with aqueous solutions prepared at 20 mg/kg IVcequivalent dose and as drug in capsule formulations at doses of 20, 75,and 203 mg/kg of IVc equivalents.

In rat oral dose escalation study, Icb-03 was dosed as aqueous solutionsat doses of 4.5, 21, and 163 mg/kg of IVc equivalents. Significantimprovements in AUC and Cmax of IVc, parent compound, after oral dosingof Icb, the prodrug, were observed compared to the historical data afterIVc oral dosing.

Metabolism and Pharmacokinetics Summary

Summary of Findings and Interpretation

Icb is the phosphate prodrug of the N-hydroxymethyl adduct of IVc and ishydrolyzed by alkaline phosphatase (ALP) to form IVc. Afteradministration of Icb to animals, therefore, plasma samples wereprepared from blood collected in the presence of EDTA, a known ALPinhibitor. Conversion of Icb to IVc was minimal (<2%) in the bloodcontaining EDTA (rat, dog, monkey and human). No significant ex vivoconversion of Icb is expected during sample storage (−20° C.) andanalysis of Icb.

The hydrolysis of Icb was studied in animal and human in vitro systems.Since multiple ALP isoforms are widely distributed in various tissues,quantitative in vitro to in vivo correlations were not attempted(Fishman et al., 1968; Komoda et al., 1981; Moss, 1983; Yora andSakagishi, 1986; Sumikawa et al., 1990). Therefore, the studies werelimited to a qualitative assessment of ALP-dependent hydrolysis indifferent tissues. Icb was hydrolyzed in the presence of serum (rat,dog, monkey and human), hepatocytes (rat, dog and human) as well as inhuman placental ALP. No turnover was observed in monkey hepatocytes. Theconversion of Icb to IVc was near stoichiometric. Due to hydrolysis inserum, the protein binding of Icb could not be determined.

IVc was rapidly formed following IV administration of Icb in rats, dogsand monkeys. The IV AUC conversion ratios were 1.0 in rats, 0.67 in dogsand 0.90 in monkeys, suggesting good conversion from Icb to IVc.

Good oral bioavailabilities (80-122%) of IVc were observed afteradministration of Icb in rats, dogs and monkeys. More significantly, thehigher aqueous solubility of Icb lessened the dissolution-rate limitedabsorption of IVc below certain doses and thereby increased theexposures of IVc in oral dose escalation and toxicokinetic studies ascompared to the exposures from the historical IVc studies.

Methods

The studies described in this report used the monotromethamine salt ofIcb, unless stated otherwise.

Quantitation of Icb and IVc by LC/MS/MS

An LC/MS/MS method was developed for the analysis of Icb and IVc inplasma samples from the animal pharmacokinetic studies as well as inacetonitrile supernatant from in vitro incubation studies. For theanalysis in plasma, a Packard Multiprobe instrument was used to transfer50 μL of each standard, QC, and plasma sample to a clean 96-well platefor protein precipitation extraction. After the addition of 200 μL ofacetonitrile containing the internal standard Compound X below, thesamples were vortex mixed and the resulting supernatant was separatedfrom the precipitated proteins by centrifugation for 10 min. For theanalysis in the supernatant generated from the in vitro studies, anequal volume of the supernatant and acetonitrile containing the internalstandard was mixed. An aliquot of the above supernatant was transferredusing a Tomtec automated liquid handler to a second clean 96-well plate.An equal volume of water was added, and the plate was capped and vortexmixed.

The HPLC system consisted of Shimadzu LC10ADvp pumps (Columbia, Md.) anda HTC PAL autosampler (Leap Technologies, Cary, N.C.) linked to aPhenomenex Synergi Fusion-RP analytical column (2.0×50 mm, 5μ; Torrance,Calif.). Mobile phase A consisted of 5 mM ammonium formate in water;mobile phase B was 100% acetonitrile. LC flow rate was 0.4 mL/min. Theinitial mobile phase composition was 3% B, ramped to 60% B over 1.75 minand held for 0.5 min, ramped to 100% B over 0.1 min and held for 0.5min, returned to initial conditions over the next 0.1 min, andre-equilibrated. Total analysis time was 4.0 min. The retention time forIcb, IVc and Compound X was 1.2, 1.7 and 1.6 min, respectively.

The HPLC system was interfaced to a Sciex API4000 triple quadrupole massspectrometer (Toronto, Canada) equipped with the Turboionspray sourceset at 550° C. and the ionspray voltage set to 4.5 kV. UHP nitrogen wasused as nebulizer and auxiliary gas with the pressure of 80 psi and 7L/min, respectively. The collision energies for Icb, IVc and Compound Xwere 23, 29 and 37 volts, respectively. Data acquisition utilizedselected reaction monitoring (SRM). Ions representing the positive ionmode (M+H)⁺ species for Icb, IVc and the internal standard were selectedin MS 1 and collisionally dissociated with nitrogen and optimizedcollision energies to form specific product ions subsequently monitoredby MS2. The SRM transitions for Icb, IVc and Compound X were m/z584→486, 474→256 and 460→218, respectively.

Standard curves ranging from 5 nM to 10 μM were prepared from stocksolutions and serially diluted in matrix for both Icb and IVc. Standardcurves were aliquoted in duplicate, extracted with the samples, andinjected at the beginning, middle, and end of the analytical sequence.The standard curves were fitted with a linear regression weighted byreciprocal concentration 1/x². Data and chromatographic peaks wereprocessed and concentrations of standards and unknowns were quantitatedusing PEBiosystems Analyst™ 1.1.

In Vitro Methods

(1) Stability of Icb in EDTA Blood, Serum and Tris-HCl Buffer

The stability of Icb was studied in fresh blood and serum from rat, dog,monkey and human (n=2). The blood was collected in vacutainerscontaining K₂EDTA (Becton Dickinson, Franklin Lakes, N.J.). The serumwas collected in vacutainers containing no anticoagulant. Icb wasincubated at a starting concentration of approximately 10 μM for 90 minat 37° C. Serial samples were taken at the pre-determined times.Aliquots of blood samples (200 μL) were first mixed with 100 μL of waterfollowed by 400 μL of acetonitrile. The serum samples (50 μL) were addedinto microtainers containing K₂EDTA (Becton Dickinson, Franklin Lakes,N.J.) followed by the addition of 100 μL of acetonitrile. Thesupernatant was analyzed for both Icb and IVc by LC/MS/MS.

The stability of Icb was also evaluated, as described above, in Tris-HClbuffer (0.1 M, pH 7.5).

(2) Hydrolysis of Icb in the Presence of Human Placental ALP

Solid human placental ALP was obtained from Sigma (P-3895, St. Louis,Mo.). A solution of 1000 units/L was prepared in Tris-HCl buffer (0.1 M,pH 7.5). Solutions of 100 and 10 units/L were obtained by serialdilution. Icb was incubated in the 10, 100 and 1000 units/L solutions(n=2) at 37° C. for 2 hr. The starting concentration of Icb in theincubation was 10 μM. Aliquots of 100 μL samples were taken atpre-determined times and added into K₂EDTA microtainers followed by theaddition of 200 μL of acetonitrile. The supernatant was analyzed forboth Icb and IVc by LC/MS/MS.

(1) In Vivo Studies in the Rat

Male Sprague-Dawley rats (300-350 g, Hilltop Lab Animals, Inc.,Scottsdale, Pa.) with cannulas implanted in the jugular vein were used.The rats were fasted overnight in the PO pharmacokinetic studies. Bloodsamples were collected from the jugular vein.

In the IV study, Icb was delivered at 1.4 mg/kg (free acid, or 1.1 mg/kgof IVc equivalent) as a bolus over 0.5 min (n=3). The concentration ofthe dosing solution was 1.4 mg/mL, and the dosing volume was 1 mL/kg.Serial blood samples were collected before dosing and at 2, 10, 15, 30,45 min, 1, 2, 4, 6, 8 and 24 hr after dosing.

In the PO study, Icb was administered at 6.9 mg/kg (free acid, or 5.6mg/kg of IVc equivalent) by oral gavage (n=3). The concentration of thedosing solution was 1.4 mg/mL, and the dosing volume was 5 mL/kg. Serialblood samples were collected before dosing and at 15, 30, 45 min, 1, 2,4, 6, 8 and 24 hr after dosing.

(2) In Vivo Studies in the Dog

The IV and PO studies of Icb were conducted in a crossover fashion inthree male beagle dogs (12±2.8 kg, Marshall Farms USA Inc., North Rose,N.Y.). There was a two-week washout period between the IV and POstudies.

In the IV study, Icb was infused via the cephalic vein at 1.3 mg/kg(free acid, or 1.0 mg/kg of IVc equivalent) over 5 min at a constantrate of 0.1 mL/kg/min. The concentration of the dosing solution was 2.6mg/mL, and the dosing volume was 0.5 mL/kg. Serial blood samples werecollected from the femoral artery before dosing and at 5, 10, 15, 30, 45min, 1, 2, 4, 6, 8 and 24 hr after dosing.

In the PO study, the dogs were fasted overnight before dosing. Icb wasadministered by oral gavage at 6.0 mg/kg (free acid, or 4.9 mg/kg of IVcequivalent). The concentration of the dosing solution was 12 mg/mL, andthe dosing volume was 0.5 mL/kg. Serial blood samples were collectedbefore dosing and at 15, 30, 45 min, 1, 2, 4, 6, 8 and 24 hr afterdosing.

(3) In Vivo Studies in the Monkey

The IV and PO studies of Icb were conducted in a crossover fashion inthree male cynomolgus monkeys (10±1.6 kg, Charles River BiomedicalResearch Foundation, Houston, Tex.). There was a two-week washout periodbetween the IV and PO studies.

In the IV study, Icb was infused via the femoral vein at 1.4 mg/kg (freeacid, or 1.1 mg/kg of IVc equivalent) over 5 min at a constant rate of0.1 mL/kg/min. The concentration of the dosing solution was 2.8 mg/mL,and the dosing volume was 0.5 mL/kg. Serial blood samples were collectedfrom the femoral artery before dosing and at 5, 10, 15, 30, 45 min, 1,2, 4, 6, 8 and 24 hr after dosing.

In the PO study, the monkeys were fasted overnight before dosing. Icbwas administered by oral gavage at 4.9 mg/kg (free acid, or 4.0 mg/kg ofIVc equivalent). The concentration of the dosing solution was 9.8 mg/mL,and the dosing volume was 0.5 mL/kg. Serial blood samples were collectedbefore dosing and at 15, 30, 45 min, 1, 2, 4, 6, 8 and 24 hr afterdosing.

(4) Data Analysis

All results are expressed as mean±SD, unless specified otherwise.

The pharmacokinetic parameters of Icb and IVc were calculated byNon-Compartmental Analysis using the KINETICAT™ software program(version 4.0.2, InnaPhase Co., Philadelphia, Pa.). The Cmax and Tmaxvalues were recorded directly from experimental observations. TheAUC_(0-n) and AUC_(tot) values were calculated using the mixedlog-linear trapezoidal summations. The total body clearance (Cl), meanresidence time (MRT), and the steady state volume of distribution (Vss)were also calculated after intravenous administration. The absolute oralbioavailability (expressed as %) was estimated by taking the ratio ofdose-normalized AUC values after oral doses to those after intravenousdoses.

The in vitro intrinsic clearance of Icb in hepatocytes (Cl_(int)) wascalculated as follows:Cl_(int)(μL/min/million cells)=Rate/C _(E)where Rate is the rate of metabolism in hepatocytes (pmol/min/millioncells), and C_(E) is the concentration of Icb in the incubation.

The in vivo intrinsic hepatic clearance of Icb (Cl_(int,in vivo)) wascalculated as follows:

${C\;{l_{{{int} \cdot {in}}\mspace{14mu}{vivo}}\left( {{mL}\text{/}\min\text{/}{kg}} \right)}} = {{Cl}_{int} \times \frac{120\mspace{14mu}\left( {{million}\mspace{14mu}{cells}} \right)}{g\mspace{14mu}{liver}} \times \frac{\chi\mspace{14mu} g\mspace{14mu}{liver}}{{kg}\mspace{14mu}{body}\mspace{14mu}{weight}} \times \frac{1}{1000}}$where χ is 40, 32, 30 and 26 g liver/kg body weight for the rat, dog,monkey and human, respectively (Davis and Morris, 1993).

The hepatic clearance Cl_(H) was calculated from the following equationusing the well-stirred model:

${C\;{l_{H}\left( {{mL}\text{/}\min\text{/}{kg}} \right)}} = \frac{{Qh} \times {Cl}_{{{int} \cdot {in}}\mspace{14mu}{vivo}}}{{Qh} + {Cl}_{{{int} \cdot {in}}\mspace{14mu}{vivo}}}$where Qh is the liver blood flow of 55, 31, 44 and 21 mL/min/kg for therat, dog, monkey and human, respectively (Davis and Morris, 1993).

The hepatic extraction ration (ER) was calculated at follows:ER=Cl_(H) /QhStudent's t-test was used for statistical analysis (Microsoft® Excel,Redmond, Wash.). Differences were considered statistically significantat the level of P<0.05.In Vitro StudiesStability of Icb in EDTA Blood, Serum and Tris-HCl Buffer

As part of the analytical assay validation, the stability of Icb wasstudied in blood containing EDTA, which is known to be an inhibitor ofalkaline phosphatases (Bowers, Jr. and McComb, 1966; Yora and Sakagishi,1986). After incubation at 37° C. for 90 min, there was less than 2%conversion of Icb to IVc in blood containing EDTA and in the presence ofTris-HCl buffer (Tables 39 and 40). Under the sample storage conditionof −20° C., the above small percentages of conversion observed at 37° C.are not expected to introduce any significant ex vivo conversion duringthe analysis of Icb.

TABLE 39 Stability of Icb in the Fresh EDTA Blood from Rat, Dog andMonkey Rat Blood Dog Blood Monkey (n = 2) (n = 2) Blood (n = 2) IVc IVcIVc Time Icb Formed % IVc Icb Formed % IVc Icb Formed % IVc (min) (μM)(μM) Formed* (μM) (μM) Formed* (μM) (μM) Formed* 0 12 0.21 1.7 10 0.0870.72 12 0.26 2.2 15 11 0.26 2.2 10 0.087 0.72 12 0.17 1.4 30 15 0.37 3.19.4 0.087 0.72 11 0.16 1.4 45 14 0.38 3.2 11 0.10 0.86 12 0.19 1.6 60 140.41 3.4 11 0.10 0.84 11 0.19 1.6 90 12 0.39 3.3 11 0.11 0.91 11 0.191.5 *Percentage formed as the starting concentration of Icb

TABLE 40 Stability of Icb in the Fresh EDTA Blood from Human, andTris-HCl Buffer Human Blood Tris-HCl (n = 2) Buffer (n = 2) IVc IVc TimeFormed % IVc Formed % IVc (min) Icb (μM) (μM) Formed* Icb (μM) (μM)Formed* 0 11 0.083 0.69 11 0.17 1.5 15 11 0.088 0.74 11 0.18 1.5 30 130.091 0.76 11 0.23 1.9 45 12 0.10 0.80 11 0.24 2.0 60 11 0.092 0.76 100.26 2.2 90 11 0.091 0.76 11 0.30 2.5 *Percentage formed as the startingconcentration of Icb

To investigate the hydrolysis of Icb in the systemic circulation, Icbwas incubated in fresh serum (rat, dog, monkey and human) at 37° C. for90 min. The rate of hydrolysis was most rapid in the monkey serum,followed by rat, human and dog sera (Table 41). The conversion of Icb toIVc was near stoichiometric.

Serum contains lower ALP activities as compared to tissues (McComb etal., 1979a). In addition, serum also contains ALP isoforms from tissuesources such as bone, liver and intestine, as a result of enzyme leakagethrough the blood vessels (Moss, 1983). Therefore, the hydrolysis of Icbin serum was probably mediated by multiple isoforms of ALP.

TABLE 41 Stability of Icb in the Fresh Serum from Rat, Dog, Monkey andHuman Rat Serum Dog Serum Monkey Human Serum (n = 2) (n = 2) Serum (n =2) (n = 2) IVc IVc IVc IVc Icb Formed Icb Formed Icb Formed Icb FormedTime (min) (μM) (μM) (μM) (μM) (μM) (μM) (μM) (μM)  0 7.6 0.23 10 0.0469.3 0.15 10 0.087 15 6.5 1.4 9.7 0.52 7.1 2.4 8.8 1.2 30 4.9 3.4 9.1 1.54.5 4.8 7.7 2.7 45 4.4 5.6 8.5 2.3 3.3 7.2 6.6 3.9 60 3.4 7.1 8.1 3.02.3 8.4 5.9 4.6 90 2.1 8.7 6.9 4.0 1.1 9.6 4.7 6.1 t_(1/2) 42 156** 3089 (min)* *Calculated as the disappearance of Icb. **The half-lives aregreater than the incubation period.Hydrolysis of Icb in the Presence of Human Placental ALP

To study the hydrolysis of Icb in a purified form of human ALP, Icb wasincubated at 37° C. (2 hr) with solutions containing human placental ALP(10, 100 and 1000 units/L). The disappearance t_(1/2) of Icb wasdetermined (Table 42). As expected, the rate of hydrolysis was faster inthe solutions with higher ALP activities. IVc was also formedaccordingly (FIG. 20). This indicates that Icb is hydrolyzed by the ALPderived from humans to form IVc.

TABLE 42 Hydrolysis of Icb in Human Placental ALP Solutions ALP Activity(Units/L) (n = 2) 10 100 1000 t_(1/2) (min) 250 29 2.4 Note: Icb wasincubated at a starting concentration of 10 μM at 37° C. for 120 min.In Vivo StudiesIn Vivo Studies in the Rat

The pharmacokinetic parameters of Icb and IVc in rats after IV and oraladministration of Icb are summarized in Table 43. The plasmaconcentration versus time profiles are shown in FIG. 21. For comparison,the historical data from the pharmacokinetic studies of IVc in rats arealso shown.

The total body clearance (Cl) of Icb following IV administration was 49mL/min/kg, suggesting that Icb is a high clearance compound in rats. Theelimination half-life (t_(1/2)) and mean residence time (MRT) after IVadministration were 0.084 hr and 0.072 hr, respectively. The volume ofdistribution of Icb at steady state (Vss) was 0.21 L/kg, suggesting verylimited tissue distribution. The formation of IVc from Icb after IVadministration was rapid; IVc was detected at the first sampling timepoint of 2 min (data not shown). The IV AUC ratio of IVc formed from Icbvs. from the historical IVc study was 1.0 (theoretical value forcomplete conversion=1), suggesting complete conversion of Icb to IVc.

Icb was not detected (LLQ=5 nM) in any plasma samples after oraladministration. The Tmax of IVc after oral administration of Icb was0.80 hr, which is shorter than the historical Tmax of IVc of 4.0 hr,indicating more rapid absorption of IVc following the oraladministration of the prodrug. The more rapid absorption of IVc from theprodrug is likely the result of better aqueous solubility of Icb as wellas rapid hydrolysis of Icb to form IVc in the intestine. The absoluteoral bioavailability of IVc from Icb was 80%, similar to the historicalIVc value of 82% (Table 43). Moreover, the exposure of IVc from the Icbrat oral dose escalation study was superior as compared to thehistorical data with IVc (Table 37 and FIG. 18).

The terminal plasma concentration vs. time profiles of IVc formed fromIcb are similar to the historical IVc profiles (FIG. 21).

TABLE 43 Pharmacokinetic Parameters of Icb and IVc Following IV and OralAdministration of Icb in the Rat (Mean ± SD, n = 3) IVc Formed Icb afterDosing with PK Parameters (03-002) Icb Historical IVc IV Dose (mg/kg)1.4 free acid 1 or 1.1 of IVc eqv. AUC_(tot) (μM * hr) 0.84 ± 0.24 30 ±4.1  27 ± 4.0  CL_(tot) 49 ± 12 NA 1.3 ± 0.19 (mL/min/kg) T_(1/2) (hr)0.084 ± 0.012 2.9 ± 0.14 4.3 ± 1.1  MRT (hr) 0.072 ± 0.008 NA 4.5 ± 0.77Vdss (L/kg)  0.21 ± 0.033 NA 0.36 ± 0.098 IV IVc AUC NA 1.0* NA Ratio PODose (mg/kg) 6.9 free acid 5 or 5.6 of IVc eqv. Tmax (hr) ND 0.80 ±0.30  4.0 Cmax (μM) ND  23 ± 0.60 13 ± 3.6  C-24 hr (μM) ND 0.14 ± 0.0630.19 ± 0.048 AUC_(tot) (μM * hr) ND 122 ± 17  111 ± 25  T_(1/2) (hr) ND3.1 ± 0.42 3.0 ± 0.28 Bioavailability ND 80**   82 (%) PO IVc AUC NA 0.98* NA Ratio NA—not applicable; ND—not detected (<5 nM). *The ratioswere calculated from IVc AUC after prodrug dosing/IVc AUC after IVcdosing for IV and PO, respectively. **Calculated from historical IV dataof IVcIn Vivo Studies in the Dog

The pharmacokinetic parameters of Icb and We in dogs after IV and oraladministration of Icb are summarized in Table 44. The plasmaconcentration versus time profiles are shown in FIG. 22. For comparison,the historical data from the pharmacokinetic studies of IVc in dogs arealso shown.

The Cl of Icb after W administration was 64 mL/min/kg, significantlyhigher than the liver blood flow of 31 mL/min/kg in dogs, and suggeststhe involvement of extrahepatic hydrolysis and/or other route(s) ofelimination (e.g., renal excretion). The t_(1/2) and MRT after IVadministration were 0.25 hr and 0.14 hr, respectively. Icb was notdetected beyond 2 hr. The Vss of Icb was 0.50 L/kg, suggesting lowpotential for tissue distribution. The formation of We from Icb after IVadministration was rapid; IVc was detected at the first sampling timepoint of 5 min (data not shown). The IV AUC ratio of We formed from Icbvs. from the historical IVc study was 0.67, suggesting moderateconversion of Icb to IVc in dogs after W administration.

Icb was not detected (LLQ=5 nM) in any plasma samples oraladministration. The Tmax of IVc after oral administration of Icb was0.58 hr, which is shorter than the historical Tmax of IVc of 1.3 hr,indicating more rapid absorption of IVc following the oraladministration of the prodrug. The absolute oral bioavailability of IVcfrom Icb was 104%, similar to the historical IVc data of 89%. Moreover,the exposure of We from the Icb dog tolerability study (dose escalation)was better when compared to the historical data with IVc (Table 41 andFIG. 21).

The terminal plasma concentration vs. time profiles of IVc formed fromIcb are similar to the historical IVc profiles (FIG. 22).

TABLE 44 Pharmacokinetic Parameters of Icb and IVc Following IV and OralAdministration of Icb in the Dog (Mean ± SD, n = 3) IVc Formed Icb afterDosing with PK Parameters (03-002) Icb Historical IVc IV Dose (mg/kg)1.28 free acid 1 or 1.04 of IVc eqv. AUC_(tot) (μM * hr) 0.61 ± 0.20 9.8± 3.6  14 ± 2.5 CL_(tot) 64 ± 18 NA  2.6 ± 0.46 (mL/min/kg) T_(1/2) (hr)0.25 ± 0.10  4.1 ± 0.54 4.6 ± 1.7 MRT (hr)  0.14 ± 0.021 NA 6.3 ± 1.9Vdss (L/kg)  0.50 ± 0.088 NA 0.93 ± 0.14 IV IVc AUC NA  0.67* NA RatioPO Dose (mg/kg) 6.05 free acid 5 or 4.92 of IVc eqv. Tmax (hr) ND 0.58 ±0.38  1.3 ± 0.58 Cmax (μM) ND  16 ± 3.9  9.6 ± 0.87 C-24 hr (μM) ND 0.22± 0.16  0.15 ± 0.027 AUC_(tot) (μM * hr) ND 72 ± 27  63 ± 2.4 T_(1/2)(hr) ND  4.2 ± 0.58  3.6 ± 0.042 Bioavailability ND 104**   89 ± 12 (%)PO IVc AUC NA 1.2* NA Ratio *The ratios were calculated from IVc AUCafter prodrug dosing/IVc AUC after IVc dosing for IV and PO,respectively. **Calculated from historical IV data of IVcIn Vivo Studies in the Monkey

The pharmacokinetic parameters of Icb and IVc in monkeys following IVand oral administration of Icb are summarized in Table 45. The plasmaconcentration versus time profiles are shown in FIG. 23. For comparison,the historical data from the pharmacokinetic studies of IVc in monkeysare also shown.

The Cl of Icb after IV administration was 47 mL/min/kg, similar to theliver blood flow of 44 mL/min/kg in monkeys, suggesting that Icb is ahigh clearance compound in monkeys. The t_(1/2) and MRT after IVadministration were 0.089 hr and 0.097 hr, respectively. The Vss of Icbwas 0.27 L/kg, suggesting limited tissue distribution. The formation ofIVc from Icb after IV administration was rapid; IVc was detected at thefirst sampling time point of 5 min (data not shown). The IV AUC ratio ofIVc formed from Icb vs. from the historical IVc study was 0.90,suggesting good conversion of Icb to IVc.

Icb was not detected (LLQ=5 nM) in any plasma samples after oraladministration. The Tmax of IVc after oral administration of Icb was0.92 hr, which is shorter than the historical Tmax of IVc of 2.3 hr,indicating more rapid absorption of IVc following the oraladministration of the prodrug. The absolute oral bioavailability of IVcfrom Icb was 122%, which is higher that the historical IVc data of 64%(Table 45).

The terminal plasma concentration vs. time profiles of IVc formed fromIcb are similar to the historical IVc profiles (FIG. 23).

TABLE 45 Pharmacokinetic Parameters of Icb and IVc Following IV and OralAdministration of Icb in the Monkey (Mean ± SD, n = 3) IVc Formed Icbafter Dosing with PK Parameters (03-002) Icb Historical IVc IV Dose(mg/kg) 1.4 free acid 1 or 1.1 of IVc eqv. AUC_(tot) (μM * hr) 0.87 ±0.14  5.0 ± 0.57 5.0 ± 1.0 CL_(tot)  47 ± 8.1 NA 7.5 ± 1.5 (mL/min/kg)T_(1/2) (hr) 0.089 ± 0.031  1.2 ± 0.063 0.92 ± 0.30 MRT (hr) 0.097 ±0.003 NA  0.87 ± 0.085 Vdss (L/kg)  0.27 ± 0.043 NA  0.40 ± 0.097 IV IVcAUC NA  0.90* NA Ratio PO Dose (mg/kg) 4.9 free acid 5 or 4.0 of IVceqv. Tmax (hr) ND 0.92 ± 0.14 2.3 ± 1.5 Cmax (μM) ND 9.5 ± 1.2 4.2 ± 2.6C-24 hr (μM) ND 0.007 ± 0.002 0.017 ± 0.011 AUC_(tot) (μM * hr) ND  24 ±4.6  14 ± 4.0 T_(1/2) (hr) ND  3.2 ± 0.37  3.3 ± 0.53 Bioavailability ND122**   64 ± 29 (%) PO IVc AUC NA 2.1* NA Ratio *The ratios werecalculated from IVc AUC after prodrug dosing/IVc AUC after oral dosingfor IV and PO, respectively. **Calculated from historical IV data of IVcAdditional Profiling Section 4:Additional Studies with Prodrug Ie

Prodrug Ie was dosed orally to rats using methodology similar to thatdescribed above for the other prodrugs. Following PO dosing, parentmolecule IVe was detected in the plasma.

Additional Profiling Section 5:

Additional Studies with Prodrug If

Prodrug If was dosed orally to rats using methodology similar to thatdescribed above for the other prodrugs. Following PO dosing, parentmolecule IVf was detected in the plasma.

The following Table 47 shows the data contained from oral dosing studiesin rats as described in additional profiling sections 4 and 5.

Compound dosed If Ie II″e IIe Details and structure of Compound dosed

Cmax p.o. (nM) 2960 ± 859  10058 ± 2755  4678 ± 3295 1545 ± 332  Tmaxp.o. (hr)  1.3 ± 0.66 0.58 ± 0.14 0.92 ± 0.95 3.3 ± 1.2 F (%) (calc on91 28 11 7.7 historical IV data for parent) AUC p.o. 12.5 ± 5.1   22 ±6.3 8.7 ± 2.7 6.9 ± 1.8 (μM*hr) Cp @ 24 hr p.o. None detected 53.9 (1/3rats) Not detected 8.17 (1/3 rats) (nM) CL iv. No IV No IV No IV No IV(mL/min/kg) Vss i.v. (L/kg) No IV No IV No IV No IV T1/2 p.o. (hr)  1.9± 0.66 T1/2 i.v. (hr) No IV No IV No IV No IV Note on detection ofprodrugs in plasma and other tissues. Once a salt form of a prodrug isadministered, it is understood that in the body, scrambling of the saltmay occur. However the assays used to quantitate for prodrugs in thesubject animal models detetcs by analysis the free acid of the phophate.This analyzing for example a lysine salt lab or a free acid Iac isassumed to be analyzing for the same species and is not intended toimply that the species detected was actually the lysine salt. In thisapplication, this convention applies to samples obtained from in vivostudies and samples onlyBiology

“μM” means micromolar;

“mL” means milliliter;

“μl” means microliter;

“mg” means milligram;

“DMSO” means dimethylsulfoxide

The materials and experimental procedures used to assess the anti-HIVactivity of the parent compounds which are generated from the prodrugsin vivo are described below:

Cells:

-   -   The human T cell line MT-2 and PM1 (AIDS Research and Reference        Reagent Program, National Institutes of Health) were maintained        and propagated in Medium RPMI-1640 (Invitrogen, Carlsbad,        Calif.), containing 10% fetal Bovine serum. (FBS, Sigma, St.        Louis, Mo.).        Virus:    -   Laboratory strains of HIV-1—the T-tropic strain LAI was obtained        through the AIDS Research and Reference Reagent Program,        National Institutes of Health. It was amplified in MT-2 cells        and titered using a virus yield assay (2). Detection was        achieved through use of a reverse transcriptase assay (3),        adapted for use with a Scintillation Proximity detection        protocol (1) (Amersham Biosciences, Piscataway, N.J.).        Experiment

-   1. Compounds stocks were prepared by dissolving in DMSO to 30 mM.    For dilution plates, compounds were serially diluted 3-fold into    DMSO, using 96-well polypropylene plates, so that the concentrations    were 100-fold greater than the final assay concentration. For    antiviral and cytotoxicity assays, 2 μl was added per well (1% final    DMSO concentration).

-   2. Compounds were added from dilution plates to 96 well tissue    culture plates, containing 100 μl Medium RPMI-1640, containing 10%    fetal bovine serum at a concentration of <20 μM.

-   3. For antiviral assays, cells were infected at a multiplicity of    infection of 0.005. After 1 h at 37° C., infected cells were diluted    to 200,000 cells per ml in Medium RPMI-1640, containing 10% fetal    bovine serum. 100 μl of this solution was added per well, giving a    final volume of 200 μl.

-   4. Plates were incubated at 37° C. in a humidified CO₂ incubator and    harvested after 5 days.

-   5. Viral infections were monitored by measuring reverse    transcriptase activity in the supernatants of infected wells as    described above. The percent inhibition for each compound was    calculated by quantifying the readout level in cells infected in the    presence of each compound as a percentage of that observed for cells    infected in the absence of compound and subtracting such a    determined value from 100.

-   6. An EC₅₀ provides a method for comparing the antiviral potency of    the compounds of this invention. The effective concentration for    fifty percent inhibition (EC₅₀) was calculated with the Microsoft    Excel Xlfit curve fitting software. For each compound, curves were    generated from percent inhibition calculated at 8 different    concentrations by using a four parameter logistic model (model 205).    The EC₅₀ data for the compounds is shown in Table 48.    Results

Biological Data Key for EC₅₀s

Compounds with EC50 Compounds >50 nM but not Compounds with EC₅₀s yettested at with EC₅₀s >1 μM but higher Compounds with >5 μM <5 μMconcentrations EC50 <1 μM Group C Group B Group A′ Group A

TABLE 48 Antiviral Activity of Compounds IV (Parent Molecules) CompoundCompound Compound Compound IVb IVc IVd IVa (sodium salt) (acid form)(acid form) EC₅₀-LAI A A A A

The anti-HIV activity of the prodrugs themselves are not relevant sincethe parent molecules, as shown by the studies below, are generated fromthe prodrugs in vivo and are the active ingredient and also the majorspecies in the plasma. In addition, the prodrugs may slowly convert toparents in the in vitro assays at least to a limited extent thuscomplicating interpretation of the antiviral data.

Cytotoxicity

-   1. Cytotoxicity assays were conducted with the same MT-2 cells,    using methodology well known in the art. This method has been    described in the literature (4). In brief, cells were incubated in    the presence of drug for six days, after which cell viability was    measured using a redox-active dye reduction assay. 50 μl of XTT    reagent (1 mg/ml    2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide,    10 μg/ml phenazine methosulfate dissolved in phosphate buffered    saline) was added to each well and incubated for 3 hours. Color    formation by actively respiring cells was quantitated in a plate    reader at 450 nm, and used to determine a CC₅₀. The CC50 for IVa,    IVb, IVc, and IVd parent molecules were greater than 10 μM when    measured by this method. The cytotoxicity data is a secondary screen    which shows the compounds are not nonspecifically killing the cells    which were used in the antiviral assay and provides further support    for the contention that the compounds possess antiviral activity.

Thus, in accordance with the present invention there is further provideda method of treating and a pharmaceutical composition for treating viralinfections such as HIV infection and AIDS. The treatment involvesadministering to a patient in need of such treatment a pharmaceuticalcomposition comprising a pharmaceutical carrier and atherapeutically-effective amount of a compound of the present invention.

The pharmaceutical composition may be in the form oforally-administrable suspensions or tablets; nasal sprays, sterileinjectable preparations, for example, as sterile injectable aqueous oroleagenous suspensions or suppositories.

When administered orally as a suspension, these compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may contain microcrystalline cellulose for impartingbulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweetners/flavoring agentsknown in the art. As immediate release tablets, these compositions maycontain microcrystalline cellulose, dicalcium phosphate, starch,magnesium stearate and lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants known in the art.

The injectable solutions or suspensions may be formulated according toknown art, using suitable non-toxic, parenterally-acceptable diluents orsolvents, such as mannitol, 1,3-butanediol, water, Ringer's solution orisotonic sodium chloride solution, or suitable dispersing or wetting andsuspending agents, such as sterile, bland, fixed oils, includingsynthetic mono- or diglycerides, and fatty acids, including oleic acid.

The compounds of this invention can be administered orally to humans ina dosage range of 1 to 100 mg/kg body weight in divided doses. Onepreferred dosage range is 1 to 10 mg/kg body weight orally in divideddoses. Other preferred dosage ranges are 1 to 20 mg/kg and 1 to 30 mg/kgbody weight orally in divided doses. It will be understood, however,that the specific dose level and frequency of dosage for any particularpatient may be varied and will depend upon a variety of factorsincluding the activity of the specific compound employed, the metabolicstability and length of action of that compound, the age, body weight,general health, sex, diet, mode and time of administration, rate ofexcretion, drug combination, the severity of the particular condition,and the host undergoing therapy.

What is claimed is:
 1. The compound, IIc, having the followingstructure: